KR20020070125A - Recombinant hepatitis c virus ns5b protein, and preparation process and use thereof - Google Patents

Abstract

The present invention expresses and isolates a water-soluble recombinant NS5B protein having RNA-dependent RNA polymerase activity in a host cell, wherein 24 amino acids are deleted from the C-terminus of Hepatitis C Virus (HCV) NS5B protein. And purifying, establishing an in vitro assay system to analyze its activity and activation conditions and to measure variants and activities of the 3′-X RNA that is its template. By applying the recombinant NS5B protein according to the present invention to high-throughput screening (HTS) analysis useful for in vitro cell culture of HCV and the development of antiviral agents in the absence of animal models, natural or non-naturally derived products Fast screening of large scale libraries.

Description

Recombinant Hepatitis C Virus NS5B protein, and preparation process and use etc.

The present invention relates to a recombinant Hepatitis C Virus (hereinafter referred to as "HCV") NS5B protein, and a method and use of the same. More specifically, the present invention provides RNA-dependent RNA polymerase (hereinafter referred to as "RdRp") activity, in which 24 amino acids are deleted from the C-terminal (Carboxy-terminal) of HCV NS5B protein. Water-soluble recombinant NS5B protein, gene encoding it, expression, isolation and purification thereof, in vitro assay system to establish the activity and activation conditions of the recombinant NS5B protein, its template 3'-X Measurement of the activity of each variant of RNA and screening of candidates for use as HCV proliferation inhibitors.

HCV is a major cause of hepatitis known to cause non-A or non-B hepatitis, and it has been reported to not only invade the human body to cause acute and chronic hepatitis, but also to cause cirrhosis or transition to liver cancer. Alter, HJ et al., 1989, N. Eng. J. Med. 321: 1494-1500). WHO currently estimates that about 3% of the world's population is infected with HCV, and in particular in Africa, Japan and Korea, 1.5-2% of the total population is chronically infected. The main feature that distinguishes hepatitis from HCV infection from hepatitis B virus (hereinafter referred to as "HBV") infection is that the probability of chronic hepatitis is much higher than that of HBV infection. That is, about 50% of HCV-induced non-A or non-B hepatitis develops into chronic hepatitis, and about 20% progresses to cirrhosis or liver cancer (Dienstag, JL, 1986, Semin.Liver) . Dis. 6: 67-81). Universal therapies have been developed to effectively treat chronic infections of HCV. However, as the only method, interferon-alpha alone or in combination with ribavirin has been administered. However, in recent years, it has been confirmed that there is no effect on HCV having interferon resistance, and the development of new therapeutic agents is urgent.

The HCV belongs to the flavivirus family and has a gene in the form of a positive strand RNA of about 9,600 bases, and consists of 3,010 to 3,033 amino acids in a single open reading frame. Produces polyprotein (Choo, QL et al., 1989, Science 244: 359-362; Choo, QL et al., 1991, PNAS 88: 2451-2455). HCV has a 5'-untranslated region (hereinafter referred to as "UTR") within about 340 nt which is important for protein synthesis. 3'-UTR, on the other hand, consists of a conserved site of short variable region-polypyrimidine tract-98 nt, which 98 nt is well conserved in various HCVs. The polyprotein of HCV consists of the core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B from the N-terminus. That is, the structural proteins (cores, E1 and E2), which are components of the nucleocapsid and envelope of the virus, are present on the N-terminus of the polyprotein, and the C-terminus is essential for the proliferation of HCV. Nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are located. The intracellular signal peptidase and the non-structural protein 2-3 (NS2-3) and the non-structural protein 3 (NS3), which are protease enzymes present in the virus, act on a specific site of the polyprotein. By cleaving, nine matured viral proteins are produced. Specifically, Core-E1-E2-p7-NS2 is cleaved by the protease of the host cell, NS2-NS3 is autocleavage by metalloprotease consisting of NS2 and 1/3 of NS3, The remainder is then processed by NS3 serine protease (Hijikata et al., 1991, PNAS 88: 5547-5551; Bartenschlarger et al. , 1994, J. Virol. 68: 5045-5055; Grakoui et al. , 1993, J. Virol . 67: 1385-1395; Tomei, etc., 1993, J. Virol 67:. 4017-4026).

Of these, NS5B has a well-conserved GDD motif in the RdRp of RNA viruses and is a major element of viral gene replication using minus strands replicated from positive strands as templates. It is reported that NS5B has no specificity for HCV RNA and can perform primer-dependent or primer-independent synthesis on a homopolymeric RNA template (Behren). , SE et al., 1996, EMBO J. 15: 12-22; Luo. G. et al. , 2000, J. Virol. 74: 851-863). In vitro studies of NS5B were obtained using recombinant proteins. For example, the baculovirus vector system was used to express recombinant NS5B in insect cells, indicating that NS5B has RdRp activity (Behrens, SE et al., 1996, EMBO. J. 15: 12-22). In addition, in Escherichia coli, NS5B was expressed as an inclusion body using glutathione-S-transferase (GST) fusion (Yuan, ZH et al., 1997, BBRC 232: 231-235), and further, the solubility of NS5B was increased. Recombinant NS5B having 21 amino acids having high hydrophobicity located at the C-terminus of NS5B was fused with GST, and Escherichia coli was cultured at 30 ° C. to express the fusion protein in water-soluble form (Yamacita, T. et al. , 1998, J. Biol. Chem. 273: 15479-15486). Alternatively, methods of directly expressing NS5B to obtain an inclusion body and then refolding to obtain an active protein or to isolate a very small amount of water soluble protein are known (Al, RH et al., 1998, Virus Res. 53: 141-149; Ferrari, E. et al. , 1999, J. Virol. 73: 1649-1654). However, methods have not yet been developed to express, isolate and purify high purity water soluble recombinant NS5B protein exhibiting excellent RdRp activity.

We cloned the NS5B gene from the sera of a Korean hepatitis C patient and deleted 24 amino acids from the C-terminus (hereinafter referred to as "NS5Bt"). In addition, by using an expression vector that contains a histidine tag downstream of the C-terminus of NS5Bt, the gene is expressed in an aqueous form in Escherichia coli, and is isolated and purified with high purity to provide a water-soluble protein having excellent RdRp activity. Could get In addition, we used this protein to build an in vitro assay system and identified the effects of inhibitors and substrate analogs on RdRp activity. In addition, the nucleotide sequence and activity of each variant of the 3'-X RNA that is the template for this protein were analyzed. These results can be useful for high-throughput screening (HTS) analysis of antiviral agents in the development of therapeutics for HCV infection.

Accordingly, an object of the present invention is to fusion using a water-soluble recombinant NS5B protein, a gene encoding the recombinant protein, an expression vector expressing the recombinant protein, and the expression vector. A method for preparing HCV transcriptase, and a method for analyzing the activity and activation conditions of HCV transcriptase with high accuracy using the enzyme, and measuring the activity of each variant of 3'-X RNA that is a template of HCV transcriptase. Methods and screening antiviral agents with high sensitivity and specificity by analyzing the effects of inhibitors or substrate analogs on RdRp activity.

1 is a diagram showing the results of electrophoresis of E. coli BL21 (DE3) / pET21b-NS5Bt transformed with the expression vector pET21b-NS5Bt of the present invention;

Figure 2a and 2b is a view showing the results of comparing the DNA and amino acid sequence of the recombinant NS5B protein and the known DNA and amino acid sequence of the NS5B protein according to the present invention;

3a to 3e are schematic diagrams showing the purification of recombinant NS5B protein according to the present invention, and electrophoresis results for purity and activity analysis of purified recombinant NS5B protein;

Figure 4 shows the results of electrophoresis for the analysis of the effect of inhibitors and substrate analogs on RNA-dependent RNA polymerase activity on recombinant NS5B protein according to the present invention;

5a to 5d are diagrams showing the steps of the in vitro assay system of recombinant NS5B protein and electrophoresis results for activation condition analysis according to the present invention; And,

6a and 6b show the results of electrophoresis for comparative analysis of the base sequence and activity of each variant of the template 3'-X RNA of the recombinant NS5B protein according to the present invention.

First, the present invention relates to a water-soluble NS5B recombinant protein exhibiting RdRp activity obtained from the serum of a Korean hepatitis C patient. Specifically, the NS5B recombinant protein of the present invention is characterized by the deletion of 24 C-terminal amino acids showing high hydrophobicity of NS5B in order to increase water solubility. The protein is preferably one or more motifs selected from the group consisting of ³¹⁷ G ³¹⁸ D ³¹⁹ D, ²²⁰ D ²²⁵ D, and ²⁸³ G ²⁸⁷ T ²⁹¹ N, more preferably the amino acid sequence of SEQ ID NO: 8 To have.

Second, the present invention relates to a gene encoding the recombinant NS5B protein. The gene of the present invention has, for example, the nucleotide sequence set forth in SEQ ID NO: 7.

Third, the present invention relates to an expression vector containing the gene. The expression vector according to the present invention is, for example, to produce fused HCV transcriptase in E. coli. Such expression vector preferably comprises a histidine tag downstream of the C-terminus of NS5B in order to facilitate purification of the fusion HCV transcriptase, in which case the fusion HCV transcriptase may contain histidine downstream of the C-terminus of NS5B. It becomes to include. More specifically, the expression vector is pET21b-NS5Bt.

Fourth, the present invention relates to prokaryotic cells transformed with said expression vector, for example pET21b-NS5Bt, in particular E. coli, more particularly E. coli BL21 (DE3) / pET21b-NS5Bt (Accession No .: KCTC 10167BP).

Fifth, the present invention relates to a method for producing a recombinant NS5B protein comprising culturing the prokaryotic cells to induce the expression of recombinant NS5B protein and obtaining crude cell eluate, then separating and purifying the recombinant NS5B protein therefrom. In the process according to the invention, it is preferred that the recombinant NS5B protein is first purified by histidine affinity chromatography followed by secondary purification by heparin affinity chromatography and gel filtration chromatography.

Sixth, the present invention performs gel electrophoresis analysis using the recombinant NS5B protein as HCV kinase and 3'-X RNA as a template of HCV kinase, or the recombinant NS5B protein as HCV kinase and The present invention relates to a method for analyzing activity and activation conditions of HCV transcriptase, including performing a filter binding assay using a homopolymeric RNA as a template of HCV transcriptase.

Seventh, the present invention provides a method for measuring the activity of a 3'-X RNA variant comprising using a recombinant 3'-X RNA as a template for the recombinant NS5B protein and HCV transcriptase as HCV transcriptase.

Eighthly, the present invention provides a template 3'-X RNA of a recombinant NS5B protein having the nucleotide sequence set forth in SEQ ID NO: 9.

Ninth, the present invention relates to a method for screening HCV proliferation inhibitors, comprising analyzing the effect of candidate RdRp activity inhibitors or substrate analogs on the recombinant NS5B protein. In the method according to the invention, it is preferred to carry out filter binding assays on multiwell plates coated with DEAE filters. It is also preferable to use 5,6- ³ H-UTP as an isotope.

Hereinafter, the present invention will be described in detail.

The present invention provides a water-soluble recombinant NS5B protein exhibiting RdRp activity obtained from the serum of a Korean hepatitis C patient. Specifically, in the present invention, in order to obtain the gene of the fusion HCV transcriptase, the HCV gene derived from the DNA clone of HCV isolated from the serum of a Korean hepatitis C patient as a template, and described in SEQ ID NO: 1 and SEQ ID NO: Polymerase chain reaction (PCR) is performed using a primer having a nucleotide sequence. The Korean HCV gene was found to correspond to type 1b by genotyping. In this case, the primer of SEQ ID NO: 1 is NS5BF ( Nhe I) consisting of 33 bases including a Nhe I restriction enzyme recognition site for the convenience of cloning, and includes a base sequence encoding amino acid sequences 1 to 8 of NS5B. The primer of SEQ ID NO: 2 is NS5BtC ( Xho I) consisting of 31 bases including Xho I restriction enzyme recognition site for the convenience of cloning, and exhibits high hydrophobicity of NS5B to increase the water solubility of the fusion HCV transcriptase. To delete the 24 amino acids C-terminally, the nucleotide sequences encoding amino acid sequences 562-567 of NS5B are included.

The gene of the fusion HCV transcriptase obtained by the above process is cut with restriction enzymes Nhe I and Xho I, and inserted into histine pET21b vector containing histidine downstream of the C-terminus of NS5Bt to prepare an expression vector according to the present invention. The expression vector of the present invention is named pET21b-NS5Bt, and transformed into E. coli BL21 (DE3) to prepare an E. coli transformant. The E. coli positive clone expressing the fusion HCV transcriptase showing RdRp activity is an E. coli transformant having a pET21b-NS5Bt # 5 expression vector, one of seven clones (see FIG. 1).

In the present invention, the transformant is named Escherichia coli BL21 (DE3) / pET21b-NS5Bt, and the Korean Biotechnology Research Institute Gene Bank (KCTC), located at 52, Eui-dong, Yuseong-gu, Daejeon, Korea under the Treaty of Budapest, February 5, 2002 It was deposited on a date and was given accession number KCTC 10167BP.

A nucleotide sequence of a gene cloned into the expression vector pET21b-NS5Bt # 5 of the present invention is analyzed using a nucleic acid sequence automatic analyzer. Specifically, the gene of the fusion HCV transcriptase showing RdRp activity has the nucleotide sequence of SEQ ID NO: 7.

According to the results of comparing and analyzing the DNA and amino acid sequences of NS5B known by the fusion HCV transcriptase and V. Lohmann et al . (1997, J. virol . 71: 8416-8428) according to the present invention, the nucleotide sequence is 92.7%, amino acid The sequences each exhibit 98.7% homology. In particular, motif A ( ²²⁰ DTRCF ²²⁵ D), motif B ( ²⁸² SGVLTTSCG ²⁹¹ N), motif C ( ³¹⁷ GD ³¹⁹ D), and motif D ( ³⁴² ) with base sequences that are believed to play an important role in NS5B RdRp activity and are conserved For AMTR ³⁴⁶ Y), the 5 base sequences are different but the amino acid sequences are completely identical (see FIG. 2).

In addition, the present invention provides a method for producing the fusion HCV transcriptase of the present invention using the transformant prepared above. Specifically, the E. coli transformant is cultured to induce the expression of the fusion protein, and the E. coli cells expressing the fusion protein are disrupted to obtain crude cell eluate. Since the fused HCV transcriptase prepared above contains six histidines at the C-terminus, they are separated by histidine affinity column chromatography. At this time, it is preferable to use an imidazole concentration gradient as the elution solvent. Heparin affinity chromatography is performed to increase the purity of the isolated fusion HCV transcriptase. At this time, it is preferable to use NaCl concentration gradient as the elution solvent. Furthermore, in order to purify the fused HCV transcriptase to higher purity, it is preferable to further perform gel filtration chromatography (Sephacryl S-200 column). As a result, it can be confirmed that the fusion protein of about 65 kDa size was synthesized by the preparation method of the present invention (see FIG. 3).

The RdRp activity of the isolated and purified fusion HCV transcriptase is analyzed by analyzing whether the substrate labeled with the isotope is incorporated. Specifically, α- ³² P-UTP and 5,6- ³ H-UTP were purified HCV transcriptase, 4 types of rNTP, template RNA (3'-X RNA or oligo U as primers) as isotopically labeled substrates. ₁₂ / G ₁₂ and homopolymer RNA polyA / C) containing a buffer and then reacted with gel electrophoresis analysis to determine the activity of HCV transcriptase by autoradiography, or sheet or 96-well DEAE After binding to the filter, the activity of HCV transcriptase is measured by a liquid scintillation counter (see FIGS. 3 and 5).

Enzyme specificity of the fusion HCV transcriptase by treating the inhibitors (Gliotoxin, LB80326, natural product LD0001) and substrate analogs (ddUTP, ddCTP) and RDRp activity of the isolated and purified at various concentrations and analyzing their effects Check the figure (see FIG. 4).

In addition, in the present invention, in order to obtain the gene of each variant of the 3'-X RNA that is the template of HCV transcriptase, it corresponds to the middle portion of the 3'-X sequence from HCV genomic RNA obtained from the serum of hepatitis C patients in Korea. CDNA is prepared from the primers and subjected to primary PCR, and the secondary PCR is used to amplify the 3-'X sequence using a primer including a deletion site of the 3'-X sequence. The PCR product was cloned into pcDNA3 and the specificity of the highly active 3'-X RNA was confirmed by analyzing the nucleotide sequence and activity of each variant of the 3'-X RNA, which is the template for HCV transcriptase (see Fig. 6). ).

The fusion HCV transcriptase of the present invention is applied to HTS assays useful for developing antiviral agents in the absence of in vitro cell culture and animal models of HCV in developing HCV transcriptase inhibitors as therapeutic agents for HCV. This makes it possible to screen large quantities of natural or non-naturally derived libraries.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which are intended to illustrate the invention but are not intended to limit the scope of the invention in any way.

Example 1 Synthesis of Primer for Polymerase Chain Reaction

In order to obtain the gene of the fusion HCV transcriptase according to the present invention, using a nucleic acid synthesizer (DNA synthesizer, Applied Biosystems Inc. Model 394) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and Each primer of SEQ ID NO: 6 was synthesized.

<Example 2> Cloning of the fusion HCV transcriptase gene showing RdRp activity

In order to obtain a fusion HCV transcriptase gene showing RdRp activity, HCV genomic RNA was extracted from the sera of a Korean hepatitis C patient and RT-PCR was performed on the 5'-UTR site. As a result, in order to obtain HCV DNA clones from the sera determined as positive, the following experiment was performed using this sera. First, HCV genomic RNA was isolated and subjected to RT-PCR by performing cDNA synthesis, long PCR and regular PCR, respectively, followed by HCV full-length cloning. PCR was performed using the Korean HCV DNA obtained as a template.

10 ng of the HCV DNA, 0.75 μg of NS5BF (NheI) primer, 0.75 μg NS5BtC (XhoI) 10 [mu] l of 10-fold concentrated reaction buffer and 2 [mu] m of 10 mM dNTP were added to the primer. Distilled water was added to adjust the final volume to 100 μl, and 0.4 μl of Taq polymerase (5 U / μl, Perkin-Elmer Cetus, USA) was added to prepare a reaction solution. PCR was performed using this reaction solution (95 ° C., 30 sec; 55 ° C., 30 sec; 72 30 sec) was repeated 30 times, and the nucleic acid of about 1,701 base pairs was obtained.

The nucleic acid of about 1,701 base pairs was purified, digested with Nhe I, Xho I restriction enzymes, and the fragments were purified. This fragment was cloned into the Nhe I, Xho I sites of pET21b (Novagen Cat. # 70763-3) to obtain the expression vector pET21b-NS5Bt of the present invention.

<Example 3> Determination of the base sequence of the fusion HCV transcriptase gene showing RdRp activity

The expression vector pET21b-NS5Bt gene cloned in Example 2 was prepared by using a DNA terminator cycle sequencing ready reaction kit (part # 402079, Perkin Elmer, USA) using a Taq polymerase. Decided.

Example 4 Preparation of an E. Coli Transformant Producing Fused HCV Replication Enzyme Showing RdRp Activity

The expression vector pET21b-NS5Bt was transformed into E. coli BL21 (DE3) strain (Novagen Cat. # 69387-1) to transform the strain, E. coli BL21 (DE3) / pET21b-NS5Bt that can express the fusion HCV transcriptase Got it. This strain was cultured in LB medium to induce the expression of IPTG (isopropyl-β-D-thio-galactopyranoside) to confirm that the desired protein at about 65 kDa size.

Specifically, in order to identify positive clones showing NS5B activity, expression vectors encoding several recombinant proteins subcloned to pET21b and various variants of pET21b-NS5Bt whose transcription and translation were confirmed by in vitro translation were identified. Each protein was expressed in the converted E. coli, purified by nickel agarose column chromatography, and subjected to RdRp reaction in vitro, followed by electrophoresis. The results are shown in FIG. In FIG. 1, 5Bf is an RNA synthesis after performing an in vitro RdRp reaction with a recombinant NS5B protein obtained by cloning, expressing and purifying the NS5B gene of the full-length sequence obtained from the sera of a Korean hepatitis C patient in an expression vector. INT # 1 and # 2 are negative controls to identify contamination during protein purification.In vitro RdRp reaction was performed with recombinant proteins obtained by cloning, expressing and purifying variants of the integrase gene of HIV into expression vectors. RNA synthesis was then investigated. 5Bt # 5 to # 16 are in vitro RdRp reactions with recombinant NS5B protein obtained by cloning, expressing and purifying the NS5B gene variant of the C-terminal 24 amino acids obtained from the sera of a Korean hepatitis C patient. RNA synthesis was then investigated. In vitro RdRp analysis used 3'-X RNA having the nucleotide sequence set forth in SEQ ID NO: 9 as a template, and + and-indicate that the template was added or not added in the RdRp reaction in vitro, respectively. 3'-XM was labeled by in vitro transcription using α- ³² P-UTP to confirm the size of the 3'-X RNA template. As shown in FIG. 1, all but 5Bt # 5 do not show any band or show a pattern attracted from the well, which may be due to contamination during protein purification since it also appears in INT independent of RdRp. However, 5Bt # 5 was identified as an NS5B positive clone showing RdRp activity, as the reaction time showed a pattern of pushing up from the bottom.

The transformant of the present invention, Escherichia coli BL21 (DE3) / pET21b-NS5Bt, was deposited on February 5, 2002 at the Korea Biotechnology Research Institute Gene Bank and was granted accession number KCTC 10167BP.

Example 5 Expression and Purification of Fusion HCV Replication Enzymes Showing RdRp Activity

The transformed Escherichia coli cells of Example 4 were incubated overnight at 37 ° C. in LB medium containing 100 μl / ml of ampicillin. 40 ml of the culture was added to 4 L of LB medium and the culture was continued. 37 Shaking incubation at ℃ ℃ the temperature of the shaker was lowered to 15 ℃ when the absorbance of the bacteria was about 1.0 at a wavelength of 600 nm. After 30 minutes, IPTG was added to a final concentration of 0.3 mM, incubated overnight, the culture was stopped, and bacterial cell precipitates were collected by centrifugation at 5,000 rpm for 10 minutes using a centrifuge (Backman J2-21, JA14 rotor).

To the transformed E. coli cell precipitate obtained in 4 L culture was suspended by adding about 100 ml of buffer 1 (20 mM imidazole, 10% glycerol, 0.5 M NaCl, 1 mM PMSF, 20 mM Tris, pH 7.5). Cells were disrupted by sonication in an ice bath in an ultrasonic grinder (ultrasonic homogenizer 4710: Cole-Parmer, USA) at 50% power and 50% efficiency cycle for about 2 minutes. The cell homogenate obtained by the above process was centrifuged at 10,000 rpm for 30 minutes using a high-speed centrifuge (Backman J2-21M, JA14 rotor) to obtain a supernatant in which water-soluble protein was dissolved.

The supernatant obtained above was added to a nickel agarose column (His-bound metal chelation resin, Novagen, USA) equilibrated with buffer 1 and passed through at a rate of 2 ml per minute. Subsequently, 100 ml of buffer solution 1 was flowed to the column to remove unadsorbed protein, and then 200 ml of buffer solution 1 containing imidazole having a linear gradient of 0 M to 1.0 M was added to remove the proteins adsorbed onto the resin per minute. Each 4 ml of fractions were collected by eluting at a rate of 2 ml.

Heparin agarose column (Heparin) equilibrated with buffer 2 by pooling the sample collected and diluted 5 times in buffer 2 (1 mM EDTA, 10% glycerol, 1 mM DTT, 20 mM Tris, pH 7.5). Sepharose CL-6B, Amersham Pharmacia Biotech, Sweden) and passed at a rate of 2 ml per minute. Subsequently, 100 ml of buffer solution 2 was flowed to the column to remove unadsorbed protein, and 120 ml of buffer solution 2 containing NaCl of 0 M to 1.0 M linear gradient was added to remove the proteins adsorbed onto the resin per minute. Each 4 mL fractions were collected by eluting at a rate of 2 mL.

The collected samples were pooled and added to an ultrafilter (Amicon Co, USA, MW cut-off: 10,000), concentrated to a protein concentration of 2 mg / ml or more, and SDS-polyacrylamide gel electrophoresis on the concentrated protein sample. A high purity protein was collected by performing the electrophoresis.

The results are shown in FIG. Figure 3a is a schematic representation of the expression and purification of the recombinant NS5B protein, Figure 3b is a SDS-PAGE results showing the elution profile after nickel agarose column chromatography. Here, S is the supernatant in which the water-soluble protein obtained by cell disruption and centrifugation is dissolved, F is flowthrough after loading the supernatant into the nickel agarose column, and P is cell disruption and centrifugation. The precipitate obtained afterwards is shown, respectively. Peaks were seen at 100 mM imidazole concentration. 3C is an SDS-PAGE result showing the elution profile of heparin column chromatography. Here, S is a sample loaded into a heparin column diluted with a NaCl concentration of 100 mM by pooling fractions 7 to 26 eluted by a nickel agarose column, and F is a flowthrough after loading the sample onto a heparin column. Represent each. Peaks were seen at 800 mM NaCl concentration. Figure 3d is a SDS-PAGE results for comparing and measuring the purity of each step protein. Here, M denotes the size of each protein as kDa on the left side as a marker. T is the cell eluate by cell disruption, S is the supernatant in which the water-soluble protein obtained by cell disruption and centrifugation is dissolved, and Ni-NTA E is 100 mM imidazole which shows the elution peak during nickel agarose column chromatography. The concentration fraction is a sample in which heparin E is obtained by pooling the eluate obtained by heparin column chromatography. As a result of measuring the purity of each sample using a densitometer, it was found that Ni-NTA E had 54% purity and heparin E had 82% purity. In addition, Figure 3e is an electrophoresis result showing the RdRp activity of each step protein. Here, the total lysate is the supernatant in which the water-soluble protein obtained by cell disruption and centrifugation is dissolved, the Ni-NTA peak is the 100 mM imidazole concentration fraction representing the elution peak during nickel agarose column chromatography, and the heparin peak is The 800 mM NaCl concentration fraction showing the elution peak during heparin column chromatography was analyzed by in vitro RdRp analysis of the heparin eluate obtained by pooling the concentrated eluate obtained by heparin column chromatography, and PC was a positive control. Contamination by other proteins or RNA was observed up to the total lysate and Ni-NTA peak, but no contamination was observed in the heparin eluate and the RdRp activity was very good.

<Example 6> Activity measurement of the fusion HCV transcriptase showing RdRp activity

The RdRp activity of the fusion HCV transcriptase isolated and purified in Example 5 was confirmed by analyzing whether the substrate labeled with the isotope is inserted. The isotopically labeled substrate used was 10 μCi of α- ³² P-UTP, purified HCV replication enzyme, four rNTPs (0.5 mM ATP, GTP, CTP, 10 μM UTP each), 0.5 μg template 3 ′. -X RNA and Buffer 3 (20 mM Tris, pH 7.0, 5 mM MgCl ₂ , 10 mM KCl, 10 mM NaCl, 1 mM EDTA, 1 mM DTT, 20 U Rnasin, 50 μg / ml Actinomycin D) After 2 hours of reaction at 30 ° C., denatured PAGE electrophoresis was performed to determine the activity of HCV transcriptase by radiograph.

Example 7 Construction of In Vitro Assay System Using Fused HCV Replication Enzyme Showing RdRp Activity

In Example 6, in order to construct an in vitro assay system capable of rapidly screening a large amount of natural or non-naturally derived libraries using a fusion HCV transcriptase showing RdRp activity, a homo instead of a template 3'-X RNA Using polymer RNA (polyA / C, homopolymer RNA containing primers U ₁₂ / G ₁₂ as primers), HCV replication by filter binding assay, instead of measuring HCV transcriptase activity by radiograph after denaturation PAGE electrophoresis The activity of the enzyme was measured. 1 μCi of 5,6- ³ H-UTP is used as an isotopically labeled substrate, and 0.4 μg / μl of homopolymer RNA containing oligo U ₁₂ / G ₁₂ , 4 pmole / μl of primer, as template RNA. The reaction was carried out in the same manner as in Example 6, except that poly A / C was used. However, after detection of RdRp activity, HCV transcriptase activity was measured using a liquid scintillation counter after binding to sheet- and 96-well DEAE filters.

5A shows a step diagram of a method for screening antiviral agents at high speed in an in vitro RdRp assay system of Korean HCV NS5B. In addition, cpm values according to reaction time were performed by RdRp analysis using α- ³² P-UTP and 5,6- ³ H-UTP as substrates and polyA / oligo U ₁₂ as templates / primers, respectively, in FIG. 5B. Indicated. In both cases, the S / N ratio increased more than 10-fold after about 3 hours of reaction. In addition, using 5,6- ³ H-UTP as a substrate and poly A / oligo U ₁₂ as a template / primer in Figure 5c, the amount of enzyme was changed to 0.5 µg and 5 µg and the reaction temperature was 30 ° C and RdRp analysis was performed at 37 ° C., and the cpm value according to the reaction time was shown. Considering that the purity of the enzyme is 50%, it can be seen that the reaction is sensitive to the amount of enzyme and that the enzyme should be used in μg. When the reaction temperature is 37 ℃, the reaction proceeds quickly at first, but after 6 hours It can be seen that the loss of RdRp activity. In addition, RdRp analysis was performed using 5,6- ³ H-UTP as a substrate and polyA / oligo U ₁₂ as a template / primer in FIG. 5D, and detected in DEAE sheet and 96-well forms, respectively. The patterns of cpm values were compared and shown. In both cases, there was no difference, and it was concluded that using 96-well type allows for faster screening and is easier.

Example 8 Effect Analysis of Inhibitors and Substrate Analogs on RdRp Activity

In order to analyze the effect of inhibitors and substrate analogs on RdRp activity using fused HCV transcriptase showing RdRp activity in Example 6, inhibitors (Gliotoxin, LB80326, natural product LD0001) and substrate on RdRp activity of HCV transcriptase Analogs (ddUTP, ddCTP) were treated at various concentrations by diluting 1/10 of each RdRp reaction and the activity of HCV transcriptase was measured. The results are shown in Table 1 and FIG.

(PPi: pyrophosphate; PAA: phosphonoacetic acid; PFA: phosphonoformic acid; MPA: mycophenolic acid; Cer: cerulenin)

The result of Table 1 shows the cpm value detected in% when the compound of the concentration was treated when the cpm value detected after the reaction without the compound was 100% (% activity). Results for GDP to Cer are inhibitory effects of inhibitors and substrate analogs published by Lohmann et al. (2000, J. Viral Hepatitis 7: 167-174), and results for ddCTP to LD0001 are shown to be inhibitors and substrate analogs identified in the present invention. It is an inhibitory effect. On the other hand, LB80326 is a diketo acid derivative having an inhibitory effect (IC ₅₀ = 0.14 μM) has already been identified (PCT WO 00/06529) and LD0001 is a kind of natural product.

4 is a result of electrophoresis qualitatively showing the effect of inhibiting RdRp when treated with a compound of the corresponding concentration.

< Example 9> Cloning of highly active template 3'-X RNA gene

In order to obtain the gene of each variant of 3'-X RNA that is the template of HCV transcriptase, primer 3 (3 'corresponding to the middle part of the 3'-X sequence from HCV genomic RNA obtained from the sera of hepatitis C patient in Korea) -X58R) cDNA was prepared and primary PCR was performed using primer 4 (99-8939F) and primer 3 using this cDNA as a template. Using the PCR product as a template, secondary PCR was performed with primers 5 (99-9029F) and primers 6 (3'-X long primer). Each PCR was performed in the same manner as in Example 2 except for the primers used. This PCR product was digested with Hind III and Xba I and cloned into pcDNA3 treated with the same restriction enzyme and named pcDNA3-3'-X. As a result of analyzing the sequence, it was found to have the same or almost similar sequence as the 3'-X. In addition, it was confirmed that a highly active 3'-X RNA template can be obtained.

Figure 6a is an electrophoresis result showing the activity of each variant of the 3'-X RNA. RdRp analysis using each variant of the 3'-X RNA as a template showed that the band densities of # 1, 4, 5 and 7 were low and the band densities of # 6, 8, 9 and 10 were high. The # 6 variant showed the highest activity, which showed better activity than the positive control 3'-X RNA (obtained from Pohang University). Figure 6b shows the nucleotide sequence of each variant of 3'-X RNA showing activity. Low activity # 1, 4, and 7 are 3'-X RNA with short polyU (U / C) tracks, and high activity # 6, 8, and 9 are polyU (U / C). C) It was confirmed that the track was a long 3'-X RNA. What is unique is that although the length is almost similar, the activity of # 5 is weak while the activity of # 10 is strong. This seems to be because the X region, which is a conserved region of 3'-X RNA, is different by 3 bases in case of # 5, and there are many Cs, while in # 10, the conserved regions of 3'-X RNA are exactly the same and there are many T's.

The fusion HCV transcriptase of the present invention is a high-purity, water-soluble NS5B recombinant protein exhibiting excellent RdRp activity derived from the serum of hepatitis C patients in Korea, which is useful for the development of antiviral agents in the absence of in vitro cell culture and animal models of HCV. Applied to HTS assays, it allows for the rapid screening of large scale candidates that can be used as HCV proliferation inhibitors.

Claims (17)

A water-soluble recombinant NS5B protein having RNA-dependent RNA polymerase (RdRp) activity, wherein 24 amino acids are deleted from the C-terminus of HCV (Hepatitis C Virus) NS5B protein. The recombinant NS5B protein of claim 1, wherein at least one motif selected from the group consisting of ³¹⁷ G ³¹⁸ D ³¹⁹ D, ²²⁰ D ²²⁵ D, and ²⁸³ G ²⁸⁷ T ²⁹¹ N is conserved. The recombinant NS5B protein of claim 2 having the amino acid sequence set forth in SEQ ID NO: 8. A gene encoding a recombinant NS5B protein according to any one of claims 1 to 3. The gene according to claim 4, which has a nucleotide sequence set forth in SEQ ID NO: 7. An expression vector containing the gene according to claim 4. The expression vector pET21b-NS5Bt. Prokaryotic cells transformed with the expression vector according to claim 6. The Escherichia coli BL21 (DE3) / pET21b-NS5Bt (Accession No .: KCTC 10167BP) according to claim 8, transformed with the expression vector according to claim 6. A method for producing a recombinant NS5B protein comprising culturing the prokaryotic cell according to claim 8 to induce the expression of recombinant NS5B protein and obtaining crude cell eluate, thereafter separating and purifying the recombinant NS5B protein therefrom. The method for preparing a recombinant NS5B protein according to claim 10, wherein the recombinant NS5B protein is first purified by histidine affinity chromatography and then secondly purified by heparin affinity chromatography and gel filtration chromatography. Perform gel electrophoresis analysis using the recombinant NS5B protein according to claim 1 as HCV transcriptase and 3'-X RNA as a template for HCV transcriptase, or replicate the recombinant NS5B protein and HCV according to claim 1 as HCV transcriptase. A method of assaying activity and activation conditions of HCV transcriptase, comprising performing a filter binding assay using homopolymer RNA as an enzyme template. A method for measuring activity of a 3'-X RNA variant, comprising using a recombinant NS5B protein according to claim 1 as a HCV transcriptase and a variant of 3'-X RNA as a template for HCV transcriptase. Template 3'-X RNA of recombinant NS5B protein having the nucleotide sequence set forth in SEQ ID NO: 9. A method for screening an HCV proliferation inhibitor, comprising analyzing the effect of an inhibitor or substrate analog of a candidate on HCV NS5B RdRp activity on the recombinant NS5B protein according to claim 1. 16. The method of claim 15, wherein the filter binding assay is performed on a multiwell plate coated with a DEAE filter. The method of screening HCV proliferation inhibitor according to claim 15, wherein 5,6- ³ H-UTP is used as an isotope.