KR100202284B1 - Rnaseh enzymatic protein from polymerase of hbv and process of it - Google Patents

Abstract

The present invention provides a recombinant protein exhibiting RNase H enzyme activity, which is part of a polymerase (P) involved in the proliferation of human hepatitis B virus, an expression plasmid capable of producing E. coli and an RNase H enzyme protein using the present expression plasmid. In the present invention, since the RNase H enzyme protein of the present invention is stable and easy to separate and purify, including histidine labeling and the like, it can be widely used for selecting various virus growth inhibitors and RNase H enzyme inhibitors.

Description

RNase H enzyme protein derived from polymerase of human hepatitis B virus and preparation method thereof

Figure 1 shows the preparation of the expression plasmid pMPRL to produce the RNase H enzyme protein derived from HBV polymerase.

Figure 2 shows the preparation of expression plasmid pMRH derived from HBV polymerase and producing histidine labeled RNase H enzyme protein.

Figure 3 shows the results of SDS-polyacrylamide gel electrophoresis by separating and purifying the RNase H enzyme protein produced by E. coli NM522 transformed with the expression plasmid pMRH of the present invention.

Lane 1: standard protein (proteins of 97, 68, 43 and 29 kDa size, respectively),

Lane 2: crude cell eluate of E. coli transformants,

Lane 3: RNase H enzyme protein first purified on an amylose column,

Lane 4: RNase H enzyme protein purified second by histidine labeled affinity column.

Figure 4 shows the results of Western blot separation and purification of the RNase H enzyme protein produced by E. coli NM522 transformed with the expression plasmid pMRH of the present invention.

A. Results using anti-maltose binding protein,

B. Results Using Histidine Labeled ( ^MRGS HIS) Antibodies,

Each lane is as described with reference to FIG. 3.

Figure 5 shows a schematic representation of the RNA-DNA complex used to analyze the activity of the RNase H enzyme protein of the present invention.

Figure 6 shows the activity of RNase H enzyme protein produced by E. coli transformed with the expression plasmid of the present invention purified from maltose binding protein, crude cell eluate of E. coli transformant, protein expressed from expression vector pMPRL into amylose column. Proteins, proteins expressed from the expression vector pMRH were first purified on an amylose column and second purified on a histidine affinity column, and by Moroni leukemia virus (MuLV) reverse transcriptase.

Figure 7 shows the aspect of the activity of the RNase H enzyme protein of the present invention increases with the amount of the enzyme protein.

Figure 8 shows that the activity of the RNase H enzyme protein of the present invention increases with the reaction time of the enzyme protein.

Figure 9 shows the activity of the RNase H enzyme protein of the present invention is changed depending on the temperature at which the enzyme protein was reacted.

Figure 10 shows the aspect of the activity of the RNase H enzyme protein of the present invention changes depending on the pH of the enzyme protein reaction.

Figure 11 shows the activity of the RNase H enzyme protein of the present invention is changed depending on the concentration of potassium chloride.

Figure 12 shows the activity of the RNase H enzyme protein of the present invention is changed depending on the concentration of magnesium ions.

Figure 13 shows the activity of the RNase H enzyme protein of the present invention changes depending on the concentration of manganese ions.

None.

[Objective of the invention]

The present invention relates to an RNase H enzyme protein derived from a polymerase of human hepatitis B virus and a method for producing the same.

More specifically, the present invention is a recombinant protein showing RNase H enzyme activity, which is part of a polymerase (P) involved in the proliferation of human hepatitis B virus (hereinafter referred to as HBV), in E. coli An expression plasmid that can be produced and a method for producing an RNase H enzyme protein in E. coli using the present expression plasmid, wherein the RNase H enzyme protein of the present invention is stable including a histidine label and is easily separated and purified. Therefore, it can be widely used for screening various virus growth inhibitors and RNase H enzyme inhibitors.

[Technical Fields of the Invention and Prior Art of the Fields]

Hepatitis B virus is the main pathogen of hepatitis, which infects about 300 million people worldwide. It is said to invade humans and cause acute or chronic hepatitis, as well as the transition to cirrhosis or liver cancer (Tiollais and Buenda, Scientific America, 264: 48-54, 1991; Blumberg, BS, Background and perspective in advances in hepatitis research (FV Chisari, ed.) New York: Mason publishing, 1984). Much research has been conducted on HBV due to its association with liver disease and molecular biological characteristics of the virus.

HBV is a DNA virus with a coating belonging to the Hepadnavirus family, and has a spherical microstructure composed of nucleocapsid and core. The HBV genome has a DNA genome that is partly double stranded, not circular, in 3.2 kb size, with multiple genes overlapping. Specifically, the genome of HBV is polymerase gene (P; polymerase), surface antigen gene (S; surface protein; pre-S1, pre-S2, S), central antigen gene (C; core protein; pre-C, C) And four genes, the X protein gene. Among them, polymerase, surface antigen, and central antigen genes express structural proteins, and X protein genes are known to express regulatory proteins. Among them, the polymerase gene accounts for 80% of the entire viral genome and produces a 94kD protein consisting of 845 amino acids.

In HBV, surface antigen proteins outside virions are recognized by hepatocyte specific receptors and enter into hepatocytes. At this time, the rest of the incomplete double helix DNA is synthesized by HBV polymerase activity to complete the HBV DNA genome. The completed HBV DNA genome is approximately 3.5 kb of pre-genomic mRNA, central antigen (C), surface antigen (S), It produces mRNA such as regulatory protein (X). Viral proteins are made from these mRNAs, wherein the polymerase protein forms a template for synthesizing the viral genome by its reverse transcriptase activity, and forms a structure called a replicatisome with a central antigen protein, a genome mRNA and the like. This is called encapsidation, and the polymerase protein is easily encapsidated by nucleic acid affinity at a site where glutamic acid is repeated at the 3′-end. When replicasomes are formed, negative DNA chains are synthesized by the reverse transcriptase activity of HBV polymerase protein, and positive DNA chains are synthesized by DNA dependent DNA polymerase activity, from which genome mRNAs are produced. This process is repeated to maintain more than 200-300 genomic DNA pools in the cell and to express various viral proteins (Tiollais and Buenda, Scientific American, 264: 48-54, 1991; Ganem, D. and Varmus). , HE, Annu. Rev. Biochem., 56: 651-693, 1987).

Although HBV is a DNA virus, it is very characteristic to replicate the genome through reverse transcription. Viruses known to undergo reverse transcription include retroviruses, which are multifunction enzymes that simultaneously function as DNA polymerase, RNase H enzyme and nucleic acid. Known. Specifically, HBV polymerase also contains a set of functions necessary for the viral genome to replicate. Ie) protein primer, ii) RNA dependent DNA polymerase (RT), iii) DNA dependent DNA polymerase (DDDP), iii) RNA degradation Enzyme (RNase H) function and the like are present in one polypeptide. Kaplan et al. Described the reverse transcriptase activity of HBV polymerase for the first time, and many studies on the replication mechanism of HBV have been made.

As shown above, reverse transcriptase has a characteristic of having an RNase H enzyme active site in common, and these RNase H enzyme sites recognize RNA-DNA complexes and the like to selectively hydrolyze only RNA strands. Reverse transcriptase is an essential function for the reverse transcriptase action, since the RNA template can be continuously synthesized only when the RNA template is removed by the RNase H enzyme activity. This RNase H enzyme was recently known as part of the retroviral reverse transcriptase, but in fact RNase H enzyme was first discovered in the calf thymus by Hansen and Stein and subsequently continued in various prokaryotes and eukaryotes. And have been reported (Stein, Hans and Hausen, P., Science, 166: 393-395, 1969).

In general, the RNase H enzyme active site present in the HBV polymerase is present at the amino terminus of the HBV polymerase, and is highly similar to that of the Moloney murine leukemia virus (MuLV) in its amino acid sequence and nucleotide sequence. Similar has been reported (Radziwill, et al., Journal of Virology, 64 (2): 613-620, 1990). In addition, the RNase H enzyme active site is known to form a 5'-terminal oligomer of pregenomic RNA that functions as a plus strand primer by removing the pregenomic RNA. Indeed, experiments analyzing mutations have shown that the conserved sequence in the RNase H enzyme protein is essential for the virus to grow. In addition, mutation analysis using the RNase H enzyme active site of duck HBV polymerase confirmed that the RNase H enzyme site inhibited the synthesis of minus strand DNA as well as plus strand DNA, and was involved in the process of RNA packaging. However, it has recently been found that duck HBV polymerase cannot recognize the binding region ε present in the pregenomic RNA of human HBV.

Therefore, in order to understand the mechanism of action of human HBV and its polymerase, in addition to inferring indirectly through research on duck HBV, a direct study using human HBV polymerase and its RNase H enzyme protein is required. So far, human HBV has been studied relatively actively only with the surface antigen necessary to develop its vaccine and X protein, which acts as a transcriptional regulator protein during the transition to liver cancer. However, little research has been carried out on HBV polymerase, which may be an important goal in developing therapeutics for hepatitis B. This is because HBV polymerase is so strongly bound to viral particles that it is difficult to isolate and obtain a low amount of active polymerase (Radziwill, G. et al., Virology, 163: 123-). 132, 1988). Therefore, in order to develop a novel hepatitis therapeutic agent, a method of selecting a virus proliferation inhibitor using a cell line infected with HBV or the like has been mainly used. This method requires a longer time and costs more than HBV polymerase or a method using the RNase H enzyme active site directly for its development, and thus no effective therapeutic agent has been developed.

Thus, since the polymerase and the RNase H enzyme derived therefrom take a large part in the study of HBV, studies for mass production of these proteins have been continuously attempted. Recently, the present inventors have applied for a patent by expressing and separating recombinant HBV polymerase protein from E. coli and measuring its enzyme activity (see Korean Patent Application Nos. 94-3918 and 96-33998). The recombinant polymerase protein obtained above is produced in Escherichia coli in the form of a maltose binding protein (MBP) at the N-terminus or a histidine-labeled fusion protein, and maltose binding protein and histidine affinity column chromatography. It can be easily separated and purified through. However, up to now, no studies have been conducted on the RNase H enzyme active site in HBV polymerase independently using the recombinant method.

Accordingly, the present inventors are located at the 3'-end of the polymerase gene by a gene recombination method to produce an RNase H enzyme protein derived from HBV polymerase, which can be used to develop an effective hepatitis therapeutic agent, as an independent protein having activity. Obtained RNase H enzyme gene to prepare an expression plasmid comprising the same, using this expression plasmid mass production of RNase H enzyme protein in E. coli in the form of a fusion protein, separated and purified by amylose column and histidine-labeled affinity column The present invention has been completed by independently preparing the RNase H enzyme protein, which is part of the HBV polymerase.

[Technical Challenges to Invent]

An object of the present invention is to provide an RNase H enzyme protein derived from the polymerase of hepatitis B virus. The present invention provides RNase H enzyme protein present at the amino terminus of human HBV polymerase.

Specifically, the present invention provides RNase H enzyme protein in the form of a fusion protein bound to maltose binding protein. The present invention also provides a RNase H enzyme protein labeled with histidine at the carboxy terminus of the fusion protein.

It is also an object of the present invention to provide an expression plasmid for producing RNase H enzyme protein of HBV polymerase in Escherichia coli.

Specifically, the present invention provides an expression plasmid pMPRL that produces RNase H enzyme protein of HBV polymerase in the form of a maltose binding protein and a fusion protein.

The present invention also provides an expression plasmid pMRH which produces RNase H enzyme protein of HBV polymerase in the form of a fusion protein labeled with histidine and bound with maltose binding protein.

It is also an object of the present invention to provide a method for preparing RNase H enzyme protein of HBV polymerase from the transformant. Specifically, the present invention is to culture the E. coli transformant to induce the expression of the RNase H enzyme protein, and then obtain the coarse cell eluate thereof to perform amylose affinity chromatography, and the protease factor Xa to treat the enzyme protein It provides a method of manufacturing.

In addition, the present invention separates and purifies the RNase enzyme protein of high purity HBV polymerase whose activity is increased by performing histidine-labeled affinity chromatography.

In addition, the RNase H enzyme protein of the HBV polymerase obtained in the present invention can be used to select a proliferation inhibitor and RNase H enzyme inhibitor of hepatitis B virus, the application of this can develop an effective hepatitis therapeutic agent.

Composition and Action of the Invention

The present invention provides a new RNase H enzyme protein derived from the polymerase of hepatitis B virus. The RNase H enzyme protein of the present invention is a revolutionary new enzyme protein derived from human HBV polymerase, and any RNase H enzyme protein derived from human HBV polymerase may be included in the scope of the present invention.

Human HBV polymerase has a site showing RNase H enzyme activity at its amino terminus. The present invention inserts the 3'-end of the human HBV polymerase gene into an expression plasmid and expresses it in E. coli RNase H of human HBV polymerase. Produce large quantities of enzyme proteins.

The present invention provides an expression plasmid that produces RNase H enzyme protein derived from HBV polymerase in Escherichia coli.

The present invention provides an expression plasmid pMPRL capable of producing the RNase H enzyme protein in the form of a fusion protein combined with a maltose binding protein (MBP). Specifically, the gene of the RNase H enzyme subdomain derived from the HBV polymerase is obtained by performing a polymerase chain reaction (PCR). At this time, using oligonucleotides having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 as a primer (see SEQ ID NO: 1 and SEQ ID NO: 2), and expressing the HBV polymerase protein in the form of fusion of maltose binding protein as a template An existing expression plasmid pMPLX (Korean Patent Application No. 94-3918) is used. The expression plasmid pMPRL was prepared by inserting the RNase H enzyme gene amplified above into the plasmid vector pMAL-c2 (see FIG. 1).

E. coli was transformed with the expression plasmid pMPRL of the present invention and the transformant NM522 / pMPRL was deposited with the Korean spawn association on November 29, 1996 (Accession No .: KCCM-10092).

The present invention also provides an expression plasmid pMRH capable of producing histidine-labeled RNase H enzyme protein in the form of a fusion protein bound with maltose binding protein. Specifically, the present invention uses the expression plasmid pMPH (Korean Patent Application No. 96-33998), which previously produces HBV polymerase protein, which is 3 'of the existing expression plasmid pMPLX (Korean Patent Application No. 94-3918). The nucleotide sequence encoding six histidine labels is inserted at the end. The present invention obtains a DNA fragment containing the nucleotide sequence corresponding to the histidine label in the expression plasmid pMPH, and inserts the DNA fragment into the expression plasmid pMPRL to prepare the expression plasmid pMRH (see Fig. 2).

E. coli was transformed with the expression plasmid pMRH of the present invention, and the transformant was deposited with the Korean spawn association on November 11, 1996 (Accession No .: KCCM-10091).

The present invention provides a method for producing an RNase H enzyme protein derived from HBV polymerase using the above-described expression plasmid and E. coli transformants.

Specifically, the present invention induces the expression of the enzyme protein by culturing E. coli transformants transformed with the above expression plasmid, and then, the E. coli cells containing the recombinant enzyme protein are disrupted to prepare crude cell eluate. The crude cell eluate is passed through amylose resin to isolate RNase H enzyme protein in the form of a fusion protein with a buffer containing maltose, and maltose binding by adding protease factor Xa to the recombinant protein obtained therefrom. Isolate RNase H enzyme protein of HBV polymerase from protein.

In addition, the histidine-labeled RNase H enzyme protein is easily separated and purified with high purity using a histidine-labeled affinity column, a metal chelating affinity column, or the like. The histidine label not only allows the expressed RNase H enzyme protein to be easily separated and purified by affinity column chromatography or the like, but also prevents the protein from being degraded, thereby maintaining the enzymatic activity of the protein in the course of purification.

The RNase H enzyme protein purified above was reconfirmed using SDS-polyacrylamide gel electrophoresis and Western blot methods (see FIGS. 3 and 4).

In order to confirm the RNA degradation activity of the RNase H enzyme protein of the present invention, an RNA-DNA complex used as a reaction substrate of the RNase H enzyme protein is prepared. At this time, the plasmid pBS-oligo in which the plasmid pBS was modified with a DNA template is used. The plasmid pBS-oligo cleaves the restriction enzyme Sma I recognition site at which the lower 102 nucleotides of the T7 promoter have been removed, and then performs an in vitro transcription reaction using T7 RNA polymerase and radiolabeled nucleotides. RNA- DNA complexes are prepared by isolating 102 nucleotides of length RNA generated using a QIAquick nucleotide removal kit (QIAGEN) and then adding a synthetic DNA oligomer (43-mer) having the nucleotide sequence of SEQ ID NO: 3. (See Sequence Table 3). The structure of the RNA-DNA complex prepared through the above process is as shown in FIG. 5.

In order to confirm the activity of the RNase H enzyme protein prepared in the present invention, the radioactivity-labeled RNA-DNA complex is reacted with the RNase H enzyme protein to react the remaining radioactivity in the supernatant using a scintillation cocktail or the like. Measure At this time, maltose binding protein is used as a control group, crude cell eluate fraction is used as a comparative group, and commercial transcriptase of commercial Moroni leuchemia virus is used. As a result, it was confirmed that the RNase H enzyme protein of the present invention derived from HBV polymerase showed about 90% of RNase H enzyme activity compared to the RNase H enzyme present in the reverse transcriptase of commercial molecular leukemia virus (FIG. 6).

In order to investigate the enzymatic properties of RNase H enzyme protein, RNase H enzyme activity under various reaction conditions is measured. Specifically, RNA-DNA complex labeled with a suitable buffer solution and radioactivity is used to measure enzyme activity according to reaction conditions.

As a result, as the amount of the enzyme protein increases, RNase H enzyme activity is higher (see FIG. 7), and it is confirmed that a reaction time of about 3 hours is required until the enzyme activity appears (see FIG. 8). In addition, the preferred reaction temperature at which the enzyme acts is 32-42 ° C. (see FIG. 9), and the preferred pH range appears relatively wide between 7.5 and 8.8 (see FIG. 10). Further, potassium chloride concentration in the reaction solution is in the range of 20-100 mM (see FIG. 11), magnesium concentration is in the range of 4-8 mM (see FIG. 12) and manganese concentration is in the range of 4-12 mM (see FIG. 13). It is confirmed that the enzyme activity is preferred.

More specifically, the RNase H enzyme protein of the present invention confirms that the optimum temperature is 37 ℃, the optimal pH is 7.9, the optimal potassium chloride concentration is 40mM, the optimal magnesium ion concentration is 4mM and the optimal manganese ion concentration is 8mM.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Preparation of Expression Plasmid pMPRL

In order to prepare an expression plasmid pMPRL capable of producing RNase H enzyme protein derived from HBV polymerase, 3'-terminal RNase H enzyme activity of the HBV polymerase gene was prepared by polymerase chain reaction (PCR). DNA fragments corresponding to the subdomains were amplified. The 5'-terminal primer used at this time has a nucleotide sequence of SEQ ID NO: 1, and the 3'-terminal primer has a nucleotide sequence of SEQ ID NO: 2 (see SEQ ID NO: 1 and SEQ ID NO: 2). As a DNA template, an existing expression plasmid pMPLX (Korean Patent Application No. 94-3918) was used, and a polymerase chain reaction was performed with the primer to obtain a large amount of DNA fragments of about 0.5 kb. These DNA fragments were ligated with the plasmid vector pMAL-c2 digested with restriction enzyme Xmn I and then digested with restriction enzyme Eco RI to isolate only the DNA fragment corresponding to the RNase H enzyme gene. This DNA fragment was again ligated with the plasmid vector pMAL-c2 cut with the restriction enzyme Eco RI to prepare an expression plasmid pMPRL having a size of 7.2 kb (see FIG. 1). The nucleotide sequence of the ligated Eco RI site was confirmed to confirm that the open reading frame of the maltose binding protein of the plasmid vector pMAL-c2 and the RNase H enzyme gene were correctly linked in order.

Example 2 Preparation of Expression Plasmid pMRH

In order to produce an expression plasmid pMRH derived from HBV polymerase and capable of producing a histidine-labeled RNase H enzyme protein, an existing expression plasmid pMPH which produces histidine-labeled HBV polymerase protein (Korean patent application) 96-33998). The expression plasmid pMPH was digested with restriction enzymes Bam HI and Hind III to obtain a DNA fragment containing the nucleotide sequence corresponding to the histidine label. The expression plasmid pMPRL, which produces the RNase H enzyme protein of the HBV polymerase prepared in Example 1, was digested with restriction enzymes Bam HI and Hind III, and then included the histidine-labeled base sequence consisting of six histidines obtained in the above procedure. DNA fragments were inserted. As a result, an expression plasmid pMRH was produced to produce a new recombinant RNase H enzyme protein labeled with histidine at the carboxy terminus of the fusion protein to which the maltose binding protein and the RNase H enzyme protein bound (see FIG. 2).

Example 3 Expression and Isolation of RNase H Enzyme Protein from HBV Polymerase

The expression plasmids prepared in Examples 1 and 2 were used to express the RNase H enzyme protein of HBV polymerase in Escherichia coli. E. coli NM522 strains were transformed with the expression plasmids pMPRL and pMRH of the present invention and incubated overnight by inoculating the transformants in 2 × YT medium. This was inoculated again by diluting the glucose (glucose rich) medium with a content of 1: 100, and when the absorbance at the wavelength of 600 nm reached 0.5, the IPTG (isopropylthiogalactoside) was added so that the final concentration was 0.5mM and further incubated at 23 ° C for 12 hours. To express the recombinant RNase H enzyme protein of the present invention.

The cell culture solution obtained above was centrifuged at 3,000 rpm for 10 minutes, and then the cell pellet was washed with 10 ml of column buffer (10 mM Tris-hydrochloric acid, pH7.4, 200 mM sodium chloride, 1 mM EDTA) and centrifuged again to suspend the pellet. The procedure of freezing and thawing (freeze and thaw) was repeated four times, and then the cells were disrupted using three sonications for 10 seconds. The crude cell eluate prepared in the above process was centrifuged at 13,000 rpm for 30 minutes at 4 ° C., and then the supernatant was separated. This process was repeated three times, and the supernatant was passed through amylose resin. The column was washed with a 50-fold column buffer solution, and then RNase H enzyme protein in the form of a fusion protein was isolated with a buffer containing 10 mM maltose. The protease factor Xa was further added to the purified protein to separate the RNase H enzyme protein of the HBV polymerase from the maltose binding protein.

In addition, since the RNase H enzyme protein produced from the expression plasmid pMRH of the present invention has a histidine label at the carboxy terminus of the protein, it was isolated and purified using a histidine labeled affinity column. Resin (Ni-NTA, QIAGEN) used in histidine-labeled affinity column was activated with sonication buffer solution (50mM sodium phosphate, pH8.0, 300mM sodium chloride), and then the activated resin was about 1 cm in diameter. To a volume of 4-5 ml. The protein sample obtained above was passed through the column at a flow rate of 0.1 ml / min and the column with wash buffer solution (50 mM sodium phosphate, pH6.0, 300 mM sodium chloride, 10% glycerol) corresponding to a volume of 100-200 times the next protein sample. Rinse enough. Next, the recombinant protein was eluted by preparing a wash buffer containing an imidazole reagent having a concentration gradient of 0.01 to 0.5 M and sequentially passing the column.

As a result, the RNase H enzyme protein of the HBV polymerase was released from the histidine-labeled affinity column at imidazole 50 mM concentration and separated and purified. The RNase H protein of purified HBV polymerase was confirmed by performing SDS-polyacrylamide gel electrophoresis and western blot (see FIGS. 3 and 4).

Example 4 Preparation of Reaction Substrates of RNase H Enzyme Proteins

In order to confirm the activity of the RNase H enzyme protein of the present invention, RNA-DNA complex used as a reaction substrate of the RNase H enzyme protein was prepared by in vitro transcription reaction using T7 RNA polymerase. As a DNA template used to prepare a run-off transcript, plasmid pBS-oligo modified with plasmid pBS was used. E. coli was transformed with the plasmid pBS-oligo, and the transformant was cultured. The plasmid pBS-oligo was isolated and purified in a large amount by alkaline lysis. In the plasmid pBS-oligo, the restriction enzyme Sma I recognition site is located at the lower 102 nucleotides of the T7 promoter.

Plasmid pBS-oligo digested with restriction enzyme Sma I was electrophoresed in 0.7% agarose gel and then eluted to use in vitro transcription reaction. In vitro transcription reactions were plasmid pBS-oligo 2 digested with 30 μl sterile distilled water, 20 μl 5 × reaction buffer (200 mM Tris-hydrochloric acid, pH7.5, 30 mM magnesium chloride, 10 mM spermidine, 50 mM sodium chloride), 10 μl DTT, Sma I 10 μl, 5 μl 2.5 mM ATP, 5 μl 2.5 mM GTP, 5 μl 2.5 mM UTP, 5 μl 0.1 mM CTP, 3 μl RNasin, 5 μl [ ³⁵ P] CTP 50 μCi, 2 μl T7 RNA polymerase containing -5 μg Next, the total reaction volume was 100 μl and reacted at 37 ° C. for 1 hour to 2 hours. Next, the 102 nucleotide length RNAs generated using the QIAquick nucleotide removal kit (QIAGEN) were isolated, 1-5 μl of these RNAs were electrophoresed on an 8M urea-6% TBE gel and then dried The radiolabel was checked by exposing to the X-ray film for 48 hours or more and developing it.

The 102 nucleotide-length RNA, whose strength of the radiolabel was confirmed, was mixed in the same mole amount as the synthetic DNA oligomer (43-mer) having the nucleotide sequence of SEQ ID NO: 3, and then heated at 70-80 ° C for 3-10 minutes. Then, by slowly dropping the temperature at room temperature, a radiolabeled RNA-DNA complex was prepared (see SEQ ID NO: 3). The structure of the RNA-DNA complex prepared through the above process is as shown in FIG. 5.

Example 5 Confirmation of Activity of RNase H Enzyme Protein

The activity of RNase H enzyme protein of HBV polymerase was confirmed using the RNase H enzyme protein isolated and purified in Example 3 and the radiolabeled RNA-DNA complex prepared in Example 4. To confirm the activity of the RNase H enzyme protein of the present invention, 1 μg of the RNase H enzyme protein of the present invention was used at 37 ° C. for 3 hours using 40 mM tris-hydrochloric acid (pH 7.9), 4 mM magnesium chloride, 40 mM potassium chloride, and RNA-DNA complex. The reaction was stopped by treatment with 50 mM EDTA. Some of the reaction solution was used to treat the same amount of 10% ice-cold TCA and reacted at 4 ° C. for 1 hour. Next, the supernatant was obtained by centrifugation at 4 ° C. for 15 minutes at 13,000 rpm, and 20 μl of the mixture was mixed with a scintillation cocktail to measure radioactivity.

As a control, the activity of RNase H enzyme protein using only maltose binding protein, which was bound to HBV polymerase RNase H enzyme protein, was confirmed under the same conditions. In addition, the crude cell eluate fraction obtained in Example 2, the reverse transcriptase of commercially available commercial Molecular Leukemia virus, and the protein expressed from the expression plasmid pMPRL were separated and purified by amylose column and expressed from the expression plasmid pMRH. Proteins were first purified on an amylose column and secondly, RNase H enzyme activity was compared in recombinant proteins isolated and purified on histidine-labeled affinity columns. As a result, the activity of the RNase H enzyme protein of the HBV polymerase was found to be about 90% of the RNase H enzyme activity present in the reverse transcriptase of the commercial Molecular Leukemia virus (see Figure 6).

Example 6 Changes in Activity of RNase H Enzyme Protein According to the Amount of Enzyme

In order to investigate the behavior of the activity of the RNase H enzyme protein changes depending on the reaction conditions, the activity of the RNase H enzyme protein of the present invention was reacted while changing the conditions. Specifically, the amount of RNase H enzyme protein of the present invention was reduced to 0 under standard reaction conditions using 40 mM tris-hydrochloric acid (pH 7.9), 4 mM magnesium chloride, 40 mM potassium chloride, 30,000 cpm RNA-DNA complex for 3 hours at 37 ° C. Enzyme activity was measured while changing to 1.6 μg. As a result, higher RNase H enzyme activity was observed as the amount of the enzyme protein increased (see FIG. 7).

Example 7 Changes in Activity of RNase H Enzyme Protein with Response Time

In the same manner as in Example 6, 40 mM tris-hydrochloric acid (pH 7.9), 4 mM magnesium chloride, 40 mM potassium chloride, 30,000 cpm RNA-DNA complex, and 1 μg RNase H enzyme protein were reacted under standard reaction conditions at 37 ° C. The reaction time which can be seen was measured. In order for the activity of the enzyme protein to appear, a reaction time of about 3 hours was required (see FIG. 8).

Example 8 Changes in Activity of RNase H Enzyme Proteins According to Temperature

In the same manner as in Example 7, 40 mM Tris-HCl (pH 7.9), 4 mM Magnesium Chloride, 40 mM Potassium Chloride, 30,000 cpm RNA-DNA Complex, and 1 μg RNase H Enzyme Protein were reacted at standard reaction conditions at 0- As a result of measuring the RNase H enzyme activity while changing in the range of 52 ° C., it was confirmed that the enzyme activity appeared strongly in a relatively wide range of 32-42 ° C. (see FIG. 9).

Example 9 Changes in Activity of RNase H Enzyme Protein According to pH

In the same manner as in Example 6, 40 mM Tris-HCl, 4 mM Magnesium Chloride, 40 mM Potassium Chloride, 30,000 cpm RNA-DNA complex, 1 μg RNase H enzyme protein were reacted under standard reaction conditions of standard reaction conditions of 3 hours and 37 ° C. As a result of measuring the RNase H enzyme activity while changing the pH in the range of 6.0 to 10.0, the enzyme activity was strong in the range of pH 7.5 to 8.8 (see Fig. 10).

Example 10 Changes in Activity of RNase H Enzyme Protein According to Concentration of Potassium Chloride

In the same manner as in Example 6, 40 mM tris-hydrochloric acid (pH 7.9), 4 mM magnesium chloride, 30,000 cpm RNA-DNA complex, and 1 μg RNase H enzyme protein were reacted under standard reaction conditions of standard reaction conditions of 3 hours and 37 ° C. As a result of measuring the RNase H enzyme activity while changing the potassium chloride concentration from 0 to 400 mM, it was confirmed that the enzyme activity appeared when the concentration of potassium chloride in the reaction solution was in the range of 20-100 mM (see FIG. 11).

Example 11 Activity Changes of RNase H Enzyme Protein According to Magnesium Concentration

In the same manner as in Example 6, 40mM tris-hydrochloric acid (pH 7.9), 40mM potassium chloride, 30,000 cpm RNA-DNA complex, and 1μg RNase H enzyme protein were reacted in the reaction solution at standard reaction conditions of reaction time of 3 hours and reaction temperature of 37 ° C. As a result of measuring the RNase H enzyme activity by changing the concentration of magnesium ion (Mg ²⁺ ), which is a divalent cation, from 0 to 50 mM, the highest RNase H enzyme activity was observed when 4-8 mM magnesium ion was present in the reaction solution. (See FIG. 12).

Example 12 Activity Changes of RNase H Enzyme Protein According to Manganese Ion Concentration

In the same manner as in Example 6, 40mM tris-hydrochloric acid (pH 7.9), 40mM potassium chloride, 30,000 cpm RNA-DNA complex, and 1μg RNase H enzyme protein were reacted in the reaction solution at standard reaction conditions of reaction time of 3 hours and reaction temperature of 37 ° C. As a result of replacing the divalent cation with manganese ions (Mn ²⁺ ), it was confirmed that the highest RNase H enzyme activity appeared when 4-12 mM manganese ions were present in the reaction solution (see FIG. 13).

[Effects of the Invention]

As shown above, the new RNase H enzyme protein derived from the polymerase of human hepatitis B virus has a temperature range of 32-42 ° C., pH 7.5-8.8, potassium chloride concentration 20-100 mM in the reaction solution, magnesium ion concentration 4-8. Enzyme activity is large in the mM range and 4-12 mM manganese ion concentration. At this time, the optimum temperature of the enzyme protein is 37 ℃, the optimum pH is 7.9, the optimum potassium chloride concentration is 40mM, the optimum magnesium ion concentration is 4mM and the optimum manganese ion concentration is 8mM.

The RNase H enzyme protein of the present invention is a novel novel RNase H enzyme protein derived from human HBV polymerase, which is produced in large quantities in E. coli, easily isolated and purified, and maintains high enzyme activity in the process. Therefore, by using the RNase H enzyme protein of the present invention, it is possible to efficiently select virus growth inhibitors and RNase H enzyme inhibitors for various uses.

In addition, the virus growth inhibitor and RNase H enzyme inhibitor selected using the RNase enzyme protein of the present invention can be widely used as a therapeutic agent for hepatitis, liver cirrhosis and liver cancer caused by viral infection.

(Sequence list)

SEQ ID NO: 1

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 1]

5'-CCCCGTTGCC CGGGAATTCC GAACAGGTCT CTGCC-3 '

(Sequence list)

SEQ ID NO: 2

Sequence length: 18

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 2]

5'-TCACGGTGGT CTCCATGC-3 '

(Sequence list)

SEQ ID NO: 3

Length of sequence: 43

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO: 3]

5'-AATTGCGTGC GAGGCGATTG GTTTGGGGCC AGAGTGGGCC AGG-3 '

Claims (11)

Novel derivatives derived from polymerases of human hepatitis B virus exhibiting activity in reaction solutions ranging from 32-42 ° C, pH 7.5-8.8, potassium chloride concentration 20-100 mM, magnesium ion concentration 4-8 mM or manganese ion concentration 4-12 mM. RNase H enzyme protein. The RNase H enzyme protein of claim 1 in the form of a fusion protein with a maltose binding protein. The RNase H enzyme protein of claim 2 comprising a histidine label at the carboxy terminus. An expression plasmid pMPRL that produces a human RNase H enzyme protein in the form of a fusion protein with maltose binding protein. E. coli transformants transformed with the expression plasmid pMPRL of claim 4 (Accession No .: KCCM-10092). An expression plasmid pMRH in the form of a fusion protein with maltose binding protein and producing a human RNase H enzyme protein comprising a histidine label at the carboxy terminus. E. coli transformants transformed with the expression plasmid pMRH of claim 6 (Accession No .: KCCM-10091). The E. coli transformant of claim 5 or 7 is cultured to induce the expression of RNase H enzyme protein, followed by amylose affinity column chromatography with crude cell eluate, and the fractions obtained are treated with protease factor Xa. A method of separating the RNase H enzyme protein of claim 1. 10. The method of claim 9, wherein in the case of culturing the E. coli transformant of claim 8, the RNase H enzyme protein of claim 1 is separated in high purity by performing histidine-labeled affinity column chromatography. Use of the RNase H enzyme protein prepared in claim 9 or 10 for the selection of RNase H enzyme inhibitors. Use of the RNase H enzyme protein prepared in claim 9 or 10 for screening proliferation inhibitors of hepatitis B virus.