KR100746991B1 - The screening method of inhibiting agent of HBV proliferation using the interaction between the core protein and surface protein of HBV - Google Patents

Abstract

The present invention relates to a method for searching for HBV growth inhibitory substance by measuring the interaction (coupling degree) between HBV (Hepatitis B Virus) core protein and surface protein by immunodetection method, and more specifically, Among the surface proteins of HBV, a fusion protein comprising a PreS region of L protein, a core protein interacting with the protein, and a method for detecting a substance that inhibits the proliferation of HBV through immunodetection to measure the interaction between the two proteins And a search kit using the same. The search method and search kit of the present invention can be usefully used to search for new HBV growth inhibitors easily and quickly at low cost.

HBV, proliferation inhibitor, search method

Description

The screening method of inhibiting agent of HBV proliferation using the interaction between the core protein and surface protein of HBV}             

1 shows a fusion protein (Trx-PreS) and a core protein (HBcAg) in which the PreS domain of L protein, which is a surface protein of Hepatitis B Virus (HBV), is connected to the C-terminus of thioredoxin. It is a schematic diagram showing the structure,

Figure 2a is a schematic diagram showing a cleavage map of the expression vector of the Trx-PreS protein,

Figure 2b is a schematic diagram showing a cleavage map of the expression vector of HBcAg protein,

3 is a schematic diagram showing the principle of measuring the interaction between Trx-PreS and HBcAg protein using an immunodetection method,

Figure 4 is a graph showing the binding specificity of the immunodetection method of the present invention.

The present invention relates to a method for searching for a proliferation inhibitor of HBV by measuring the interaction between a hepatitis B virus (HBV) core protein and a surface protein by an immunodetection method, and more specifically, to a surface of an HBV. Method of searching for a substance that inhibits the activity of HBV through a fusion protein including the PreS region of the L protein, a core protein interacting with the protein, and an immunodetection method measuring the interaction between the two proteins among the proteins and a search using the same Relates to a kit.

Hepatitis B virus (HBV) is a virus of the Hepadnaviridae strain that specifically infects the human body.

Currently, about 200 million people worldwide are carriers of HBV, and 80% of them are concentrated in the Asia-Pacific region. In Korea, 7-8% of the population is known to be infected with HBV. A Study on the Relationship between Positive Rate of HBsAg and HBs and Liver Function Tests in the Patients, Journal of the Korean Internal Medicine.

Hepatitis B has been studied with great interest in its treatment because of its severity. Although several drugs and vaccines have been developed and received good reviews in preclinical and clinical trials, few drugs have been applied as actual therapeutics, and the only treatment used to date for HBV hepatitis patients has been to rely on the human immune system. Human immune action against HBV removes HBV particles, but on the other hand destroys hepatocytes, so side effects of these immune actions can lead to fatal diseases. In addition, since these problems make it difficult to predict disease progression, it is desirable to use a combination of antiviral therapy and immunotherapy for fast and efficient hepatitis treatment.

To date, various methods such as interferon, nucleic acid derivatives or immunomodulators have been tried to treat hepatitis B virus. In addition, the recent development and use of Ara-A (Ara-A) or acyclovir (Acyclovir) is a problem that the strong toxicity. Phosphonoformate (Foscarnet), Suramin, Prostaglandin, Famciclovir, etc. are under development, but much validation is still needed. As described above, some drugs have been developed as therapeutic agents for hepatitis B. However, few drugs have satisfactory efficacy. In particular, as a method of inhibiting the proliferation of the hepatitis B virus, it inhibits the attachment of the virus to the receptor or the activity of the virus. Research has been conducted to find substances that specifically inhibit proteins and polymerases.

HBV, a hepatitis-inducing virus, is specifically infected with hepatocytes, and proteins constituting HBV are synthesized from the gene of HBV in infected cells. In the replication of hepatitis B virus, major proteins such as core protein and surface protein are made. Core protein is structural antigen crystal and surface protein is antigen crystal on the surface of virus.

Core protein synthesized in the cytoplasm combines with HBV nucleic acid to form nucleocapsid particles having a diameter of 30 nm. On the other hand, the surface protein of HBV is located on the intracellular ER lipid membranes after biosynthesis of three L, M, and S proteins from one gene (S gene), and the nucleocapsid is specific to these surface proteins expressed on the ER membrane. At the same time, the lipid membrane of the ER surrounds the HBV nucleocapsid, forming a complete HBV particle (Volker Bruss and Don Ganem, Proc. Natl. Acad. Sci. USA , 88: 1059-1063, 1991). ).

In this process, selective binding between the core protein on the surface of the HBV nucleocapsid and the surface protein, particularly the site corresponding to amino acids 1-163 of the N-terminus of the L protein named PreS, causes the formation of HBV particles. The interaction between these two proteins can be considered as a target for the development of hepatitis therapeutics that inhibit the proliferation of HBV.

In order to search for candidates of the hepatitis therapeutic agent by measuring the interaction between the core protein and the surface protein of the HBV, it is necessary to develop a mass expression of the protein, an analysis of the interaction and a measurement method thereof.

Therefore, the present inventors have expressed a method of expressing and purifying the minimum sites necessary for the function of these proteins in the core protein and the surface protein of HBV and simply measuring the interaction (binding degree) between these sites using an immunodetection method. The present invention was completed by confirming that the measurement method can be usefully used to search for substances capable of inhibiting virus proliferation by inhibiting the interaction between core protein and surface protein of HBV.

An object of the present invention is to provide a method for screening a substance capable of inhibiting the proliferation of HBV by measuring the interaction (coupling degree) between the core protein and the surface protein of HBV.

In order to achieve the above object, the present invention is a method for searching for a substance that inhibits the growth of the virus by measuring the fusion protein containing the surface protein site of HBV, core protein of HBV and the interaction (binding degree) between the two proteins To provide.

In addition, the present invention is iii) fusion protein and core protein comprising the surface protein site of HBV; Ii) primary and secondary antibodies (labeled antibody conjugates) to the core protein; And, iii) provides a kit for screening HBV growth inhibitory substances comprising a substrate solution containing a color developer.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for searching for a substance that inhibits the proliferation of a virus by measuring the fusion protein including the surface protein portion of HBV, the core protein of HBV and the interaction (binding degree) between the proteins.

Core proteins among the structural proteins of HBV synthesized in hepatitis cells combine with HBV nucleic acids to form nucleocapsid particles having a diameter of 30 nm. The surface proteins of HBV are located on the intracellular ER lipid membranes after biosynthesis of three L, M and S proteins from one gene, and the nucleocapsid is specifically bound to these surface proteins expressed on the ER membrane. At the same time, the lipid membrane of the ER surrounds the HBV nucleocapsid to form a completed HBV particle.

In this process, selective binding between the core protein on the surface of the HBV nucleocapsid and the surface protein, particularly the site corresponding to amino acids 1-163 of the N-terminus of the L protein named PreS, causes the formation of HBV particles. Done.

Therefore, a substance capable of inhibiting HBV proliferation by inhibiting the binding between the core protein and the PreS site and inhibiting the formation of viral particles can be used as a therapeutic agent for hepatitis caused by HBV.

The fusion protein comprising the surface protein of the HBV of the present invention is composed of a site that facilitates the purification of the surface protein and a surface protein site, and more preferably, the L protein of the surface protein of thioredoxin and It may consist of a PreS site. The fusion protein may have an amino acid sequence of SEQ ID NO: 8 encoded by the nucleotide sequence described in SEQ ID NO: 7 .

In addition, the core protein may include the entire core protein portion of the HBV, but more preferably, may be composed of a portion in which the pro sequence of the entire core protein sequence is deleted. The core protein may have an amino acid sequence of SEQ ID NO: 6, encoded by the nucleotide sequence represented by SEQ ID NO: 5.

In order to express the fusion protein including the PreS region of the L protein and the core protein of E. coli in the surface protein of the HBV, expression vectors pTrx-PreS and pHBcAg each containing the gene of the protein were prepared ( FIG. 2a and 2b ).

The degree of interaction between the two recombinant proteins produced by transforming the expression vector into Escherichia coli was measured by the following immunodetection method.

Searching for a substance that inhibits the proliferation of HBV of the present invention using an immunodetection method comprises the steps of: i) adsorbing a fusion protein comprising the surface protein of HBV to the container; Ii) treating the proliferation inhibitory substance of HBV; Iii) adding core protein; Iii) binding the primary antibody against the core protein in step iii); Iii) binding a secondary antibody (labeled antibody conjugate) to step iv) and developing using the same; And, i) comparing the degree of color development.

In addition, the present invention is iii) fusion protein and core protein comprising the surface protein site of HBV; Ii) a primary antibody against a core protein and a secondary antibody thereto (labeled antibody conjugate); And iii) a kit for screening a proliferation inhibitor of HBV, including a substrate solution containing a color developer. The search kit is preferably, iii) fusion protein and core protein consisting of the PreS site and thioredoxin site of L protein in the surface protein of HBV; Ii) primary and secondary antibodies (labeled antibody conjugates) to the core protein; And iii) a substrate solution containing a color developer.

In the above search kit, the secondary antibody (conjugate of the labeled antibody) may be an antibody labeled with a horseradish peroxidase (HRP), an alkaline phosphatase, a fluorescein or a dye. Can be.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of HBcAg Protein

<1-1> Expression of HBcAg Gene

PADRa1 plasmid DNA containing the gene of HBV, including DNA having the C-terminal nucleic acid binding site and the pro sequence (amino acids 30-214) among HBV core proteins (Ahn Byung-yun, Korea University) Donated from professor) as a template, and using a primer of SEQ ID NO: 1 and SEQ ID NO: 2 , 95 ° C, 1 minute by DNA Polymerization Chain Reaction (PCR); 55 ° C., 30 seconds; It synthesize | combined 25 times at 72 degreeC and the cycle of 1 minute. The amplified DNA was digested with DNA restriction enzymes of Nde I and Hin dIII, and then linked to pET21a vector DNA (Novagen, USA) digested with the same restriction enzyme using T4 DNA ligase to produce an HBcAg protein. Prepared. Analysis of the DNA sequence confirmed that the synthesized DNA sequence and the DNA were properly inserted into the expression vector.

<1-2> HBcAg Expression and Purification of Proteins

For expression and purification of HBcAg protein, the expression vector pHBcAg prepared in Example <1-1> was first introduced into BL21-Codon Plus-RIL (Stratagene), which is a type of Escherichia coli, and the transformant was transformed into ampicillin ( 50 μg / ml) and chloramphenicol (34 μg / ml) were used to shake culture the LB medium. When the absorbance of the culture medium at 600 nm reached 0.7 to 0.8, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce the expression of HBcAg protein and further cultured for 3 hours. Collected by centrifugation.

The E. coli was suspended in a buffer solution (50 mM Na phosphate, 1% Triton X-100, pH 7.4, protease inhibitor cocktail) and passed through a French press at a pressure of 16,000 psi to destroy the cell walls and centrifugation ( It was 10,000 × g, 30 minutes) to ultracentrifugation and the supernatant obtained (140,000 × g, 2 hours). The precipitate was isolated and then dissolved in buffer (50 mM Tris-HCl, 5 mM EDTA, 5% sucrose, pH 7.8) and purified by Sephacryl S-400 gel filtration (Amersham Pharmacia Biotech) chromatography. Thus prepared protein has a purity of about 90%.

Example 2 Preparation of Trx-PreS Protein

<2-1> Expression vector preparation of Trx-PreS gene

DNA containing the PreS site of the L protein, the HBV surface protein, and pADRa1 plasmid DNA containing the HBV gene (donated by Professor Ahn Byung-yoon, Korea University) were used as primers using SEQ ID NO: 3 and SEQ ID NO: 4 95 degreeC, 1 minute by polymerization chain reaction method (PCR); 55 ° C., 30 seconds; It synthesize | combined 25 times at 72 degreeC and the cycle of 1 minute. T4 DNA in pTrx vector DNA (Jin, Y. et al., Chem. Biol. 9: 157-162, 2002) was digested with DNA restriction enzymes of BamH I and Hind III and then digested with the same restriction enzyme . PTrx-PreS, an expression plasmid that was linked using ligase to produce Trx-PreS protein, was prepared. Analysis of the DNA sequence confirmed that the synthesized DNA sequence and the DNA were properly inserted into the expression vector.

<2-2> Trx-PreS Expression and Purification of Proteins

For expression and purification of the Trx-PreS protein, the expression vector pTrx-PreS was introduced into BL21 (DE3) (Novagen, USA), a strain of E. coli, and the transformant was contained at a concentration of 50 μg / ml of kanamycin at 37 ° C. The shake was cultured using the LB culture medium. When the absorbance of the culture at 600 nm reached 0.7 to 0.8, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce the expression of the protein, and further cultured for 3 hours, after which E. coli was cultured. Collected by centrifugation. The collected E. coli was suspended in a buffer solution (50 mM Na phosphate, 300 mM NaCl, pH 8.0) and passed through a French Press at a pressure of 16,000 psi to break the cell wall, followed by centrifugation (10,000 × g, 30). Insoluble materials such as cell walls were removed. The expressed protein was passed through a Hi-Trap chelating HP column (Amersham Pharmacia Biotech) filled with Ni ^2+, and the adsorbed protein was separated by concentration gradient using 500 mM imidazole buffer. The separated proteins were collected, dialyzed in a buffer solution (20 mM Tris-HCl, pH 8.0), and passed through a Mono-Q HR column (Amersham Pharmacia Biotech), and the adsorbed protein was concentrated in a concentration gradient using 500 mM NaCl buffer. Separated. Thus prepared protein has a purity of about 95%.

Example 3 Development of Search Method Using Immunodetection Method

<3-1> Immunodetection Using HBcAg Protein and Trx-PreS Protein

      The present inventors devised an immunodetection method using the interaction (coupling degree) between the HBcAg protein of HBV and the Trx-PreS protein expressing the PreS site of L protein in the surface protein.

First, 0.1 ml of 10 μg / ml Trx-PreS protein (50 mM Na phosphate, 150 mM NaCl, pH 8.0) was added to a 96 well cell culture vessel and allowed to adsorb on a plastic surface at 4 ° C. for 14 hours. The aqueous protein solution was removed. Thereafter, 0.3 ml of blocking solution (50 mM Na phosphate, 150 mM NaCl, pH 8.0, 5% skim milk powder) was added thereto, followed by treatment at room temperature for 2 hours, followed by 0.3 ml of PBST washing solution (50 mM Na phosphate, 150 mM NaCl). , pH 7.4, 0.5% Triton X-100), washed 6 times and diluted with PBS buffer (50 mM Na phosphate, 150 mM NaCl, pH 7.4) in 0.1 ml of HBcAg protein (0.3 μM) were added at room temperature for 2 hours. Reacted for a while. After removing the aqueous protein solution, the solution was washed 6 times with PBST washing solution, and the solution of 0.1 ml of HBcAg protein (Anti-HBcAg Ab, KOMA biotechnology 1/2000 dilution) solution was added and reacted at room temperature for 1 hour. After removing the aqueous solution of the solution was washed six times with PBST washing solution and 0.1 ml of a secondary antibody (anti-rabbit peroxidase conjugate, sigma, 1/2000 dilution) was added and reacted at room temperature for 1 hour to remove the aqueous solution of the antibody. After washing 6 times with PBST washing solution, 0.1 ml of OPD coloring solution was added and reacted for 2-5 minutes. Then, 0.1 ml of 2.5 M sulfuric acid was added to stop the reaction, and the absorbance of the aqueous solution was measured at 490 nm.

<3-2> Measurement of interaction between HBcAg protein and Trx-PreS protein by immunodetection

In order to confirm whether the designed immunodetection method can selectively measure the binding degree between core protein and surface protein of HBV, the Trx-PreS protein was treated by incubation with HBcAg protein and Trx protein. When HBcAg protein was treated by adsorbing (thioredoxin, thioredoxin), the color reaction by each anti-HBcAg antibody-HRP conjugate was investigated. As a result , as shown in FIG. 4 , a very weak color reaction occurs for the Trx protein, whereas a strong color reaction occurs for the Trx-PreS protein. These results show that the color reaction in the immunodetection method is due to the interaction (binding) between the HBcAg protein of HBV and the PreS site of the surface protein. The specific binding force between PreS and HBcAg can be measured by subtracting the absorbance due to the interaction of HBcAg protein and Trx protein from the interaction between HBcAg protein and Trx-PreS. Therefore, the immunodetection method using the interaction between the HBV core protein and the surface protein of the present invention can be used as a proliferation inhibitor of HBV by inhibiting the interaction (binding) between the HBcAg protein and the PreS site of the surface protein and weakening the color reaction. It can be used to search for new drug candidates.

The method for screening HBV growth inhibitory substance using the measurement of the interaction between the core protein and the surface protein of HBV of the present invention and the search kit using the same are not conventional DNA syntheses in the search for hepatitis therapeutic agents induced by HBV. It can be used as a means of screening antiviral substances targeting new targets, suggesting a new search method, and using HBV recombinant proteins rather than surviving HBV viruses, so that HBV proliferation inhibitory activity can be measured more safely. . In addition, when the screening method of the present invention is carried out on 96-well or 384-well plates, the activity of multiple compounds can be measured simultaneously, so that the proliferation inhibitory substance of HBV can be searched simply and inexpensively.

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Claims (13)

Core protein of Hepatitis B virus (HBV) The proliferation of Hepatitis B virus (HBV) was measured by measuring the interaction (coupling) between the site that facilitates the surface protein purification of HBV and the fusion protein consisting of the PreS site of the L protein of the surface protein of the HBV. How to search for inhibitory substances. delete delete The method of claim 1, wherein the site for facilitating purification of the surface protein is thiorredoxin, and a method for screening a substance that inhibits the proliferation of Hepatitis B virus (HBV). The hepatitis B virus (HBV) according to claim 1, wherein the core protein region is a protein having an amino acid sequence of SEQ ID NO: 6 , and the fusion protein is a protein having an amino acid sequence of SEQ ID NO: 8 . To search for substances that inhibit the proliferation of The method of claim 1, wherein the method for measuring the interaction is an enzyme immunodetection method (enzymeimmunoassay). Searching for a substance that inhibits the proliferation of Hepatitis B virus (HBV). Iii) adsorbing a fusion protein consisting of a site that facilitates the purification of Hepatitis B virus (HBV) surface protein and a PreS site of L protein in the surface protein of HBV; Ii) treating proliferation inhibitory candidates of Hepatitis B virus (HBV); Iii) adding core protein of Hepatitis B virus (HBV); Iii) binding the primary antibody against the core protein of step iii); Iii) binding a secondary antibody (labeled antibody conjugate) to the primary antibody conjugate of step iv) and developing it using the same; And Iii) a search method for a substance that inhibits the proliferation of hepatitis B virus (HBV), comprising the step of comparing the degree of color development. Iii) a fusion protein consisting of a site for facilitating purification of Hepatitis B virus (HBV) surface protein and a PreS site of L protein among the surface proteins of HBV; Ii) core protein of Hepatitis B virus (HBV); Iii) primary and secondary antibodies to the core protein (labeled antibody conjugates); And I) Search kits for substances that inhibit the proliferation of Hepatitis B virus (HBV), including a substrate solution containing a coloring agent. [Claim 10] The substance for inhibiting the proliferation of Hepatitis B virus (HBV) according to claim 8, wherein the site for facilitating purification of the surface protein of step iii) is thioredoxin. Search Kit of. The core protein of Hepatitis B virus (HBV) having an amino acid sequence set forth in SEQ ID NO: 6 . Figure 11 comprising a gene of SEQ ID NO: 5 encoding the protein of claim 10. Expression vector pHBcAg as cleavage map of 2b. Amino acid sequence as set forth in SEQ ID NO: 8 in which the PreS site of the L protein of the surface protein of HBV is linked to the C-terminus of thioredoxin, which facilitates the purification of Hepatitis B virus (HBV) surface protein Fusion proteins. Figure 12 comprising a gene of SEQ ID NO: 7 encoding the protein of claim 12. Expression vector pTrx-PreS shown by cleavage map of 2a.

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