KR101929245B1 - Antiviral compositions against hepatitis B virus comprising interleukin-32 as an active ingredient - Google Patents

Antiviral compositions against hepatitis B virus comprising interleukin-32 as an active ingredient Download PDF

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KR101929245B1
KR101929245B1 KR1020160160359A KR20160160359A KR101929245B1 KR 101929245 B1 KR101929245 B1 KR 101929245B1 KR 1020160160359 A KR1020160160359 A KR 1020160160359A KR 20160160359 A KR20160160359 A KR 20160160359A KR 101929245 B1 KR101929245 B1 KR 101929245B1
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hbv
expression
virus
antiviral
cells
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KR20180060639A (en
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김균환
김두현
박영민
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단디바이오사이언스 주식회사
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    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/5067—Liver cells
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
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    • C12N2730/10011—Hepadnaviridae
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    • G01N2333/54—Interleukins [IL]

Abstract

A composition for antiviral therapy against hepatitis B virus comprising as an active ingredient interleukin-32 (IL-32), an expression or activity inhibitor of HNF4α (Hepatocyte nuclear factor 4 alpha) or HNF1α (Hepatocyte nuclear factor 1 alpha) And a method for screening antiviral substances against viruses.
When the host cell is infected with hepatitis B virus, the present inventors induced secretion of IL-32 by TNF-a and IFN-y cytokines secreted from infected cells, secreted IL-32 phosphorylated ERK1 / 2 HNF4? And HNF??, Which are involved in the transcription of the viral gene, and thereby inhibit DNA replication and gene expression of the virus. Thus, IL-32-mediated hepatitis B virus And the molecular mechanism of the antiviral effect was investigated. Therefore, the antiviral activity of IL-32 against the hepatitis B virus newly identified according to the present invention provides a new understanding for the development of a therapeutic agent for the virus and can be usefully used for the development of an antiviral therapeutic agent .

Description

The present invention relates to an antiviral composition for hepatitis B virus comprising interleukin-32 as an active ingredient,

The present invention relates to an antiviral composition for hepatitis B virus, and more particularly, to an antiviral composition comprising hepatocyte nuclear factor 4 alpha (HNF4α) or hepatocyte nuclear factor 1 alpha (HNF1α) The present invention relates to a composition for antiviral treatment against hepatitis B virus and an antiviral substance screening method for the virus.

Continuous infection with hepatitis B virus (HBV) has become a global public health problem with chronic hepatitis B (CHB), liver cirrhosis, and hepatocellular carcinoma have.

It is known that the HBV genome (3.2 Kb) in the form of relaxed circular DNA (RC) is transformed into the covalently closed circular DNA (cccDNA) form in the nucleus. HBV can be obtained from four open-reading frames (PreC / C, P, preS1 / S2 / S and X), four kinds of virus proteins: polymerase, surface protein, HBx. HBV replication occurs through reverse transcription in the core capsids using HBV polymerase proteins. In the nucleus, four types of HBV RNA encoded in the cccDNA of HBV are transcribed by an HBV enhancer. Various transcription factors such as HNF (hepatocyte nuclear factor) and C / EBP (CCAAT / enhancer binding protein) are known to be involved in the generation of HBV RNA by binding to the enhancer I and II sites of HBV, Lt; RTI ID = 0.0 > transcription factors. ≪ / RTI >

To date, various in vitro and in vivo studies have shown that cytokines inhibit gene expression, replication, and onset of HBV by a variety of mechanisms. For example, host defense systems are known to modulate the life cycle of HBV against HBV. In particular, TNF-α and IFN-γ are well known as cytokines that induce antiviral responses in host defense mechanisms. Secretion of TNF- [alpha] and IFN- [gamma] is mediated by various antiviral genes through cytokine-dependent signaling such as CIAP2, MxA, and PKR. However, another mechanism study has reported that TNF-α regulates HBV RNA production and protein outer-stability, and that IFN-γ removes the protein outer membrane containing pregenomic RNA in mouse hepatocytes. It is also known that TNF-α and IFN-γ are involved in the removal of non-cytopathic viruses. In addition to TNF-α and IFN-γ, various cytokines have also been reported to exhibit antiviral effects directly or indirectly to HBV. Therefore, it is known that various cytokines contribute to the host defense system against HBV infection, but the immune mechanism of host to HBV has not yet been clarified.

On the other hand, it is known that IL-32 is a type of cytokine, which is induced by the synergistic action of TNF-α and IFN-γ in the pancreas. IL-32 is produced by a variety of epithelial cells and immune cells such as T lymphocytes, NK cells and monocytes. The IL-32 gene is located on the human chromosome 16p13.3, and is a pro-activator that activates the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- -inflammatory is known as cytokine. The IL-32 gene produces six selective splice variants after transcription. Of these variants, IL-32γ is known to be the most active form. However, the biological function of the IL-32 isoforms is not well known. According to the results of a conventional study, IL-32 has been shown to be effective against various diseases such as orientia tsutsugamushi, vesicular stomatitis virus (VSV), human immunodeficiency virus (HIV), hepatitis C virus (Immunology 2011; 132: 410-420) have been reported to be associated with various bacteria and viruses, such as human papilloma virus (HCV), human papilloma virus (HPV), and influenza virus. However, the antiviral effects of IL-32 and its direct mechanism in relation to HBV have not been elucidated.

The inventors of the present invention have conducted intensive studies in order to elucidate the factors that induce antiviral effects of TNF-α and IFN-γ and their molecular mechanisms of action in the host immune defense system against hepatitis B virus (hereinafter, HBV) As a result, IL-32 induced by TNF- [alpha] and IFN- [gamma] inhibited the expression or activity of HNF4 [alpha] or HNF1 [alpha], which is a transcription factor present in the host hepatocyte, thereby reducing the enhancer activity of hepatitis B virus, The present invention has been completed on the basis of this finding.

Accordingly, the present invention provides a pharmaceutical composition comprising B (B) or (B) as an active ingredient, an interleukin-32 (IL-32) gene or an interleukin-32 protein, which is an inhibitor of the expression or activity of HNF4α (Hepatocyte nuclear factor 1 alpha) It is intended to provide a composition for antiviral treatment against hepatitis virus.

It is another object of the present invention to provide a screening method for an antiviral substance against hepatitis B.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a method for inhibiting the expression or activity of HNF4α (Hepatocyte nuclear factor 4 alpha) or HNF1α (Hepatocyte nuclear factor 1 alpha) or an interleukin-32 (IL- 32 protein as an active ingredient. The present invention also provides a composition for antiviral therapy against hepatitis B virus.

In one embodiment of the present invention, the interleukin-32 gene may be a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 to 4.

In another embodiment of the present invention, the interleukin-32 protein may be an amino acid sequence selected from the group consisting of SEQ ID NOS: 5 to 8.

In another embodiment of the present invention, the interleukin-32 may inhibit the expression or activity of HNF4? Or HNF1? By increasing phosphorylation of intracellular ERK1 / 2 protein.

In another embodiment of the present invention, the composition inhibits the expression or activity of HNF4 [alpha] or HNF1 [alpha], thereby reducing the enhancer activity of hepatitis B virus and inhibiting viral replication.

In addition, the present invention provides a method for screening an antiviral substance against hepatitis B, comprising the steps of:

(a) treating the candidate material in cells in vitro ;

(b) measuring the level of IL-32 expression in said cells; And

(c) selecting an antiviral substance against hepatitis B as a substance that increases the expression of IL-32 as compared to a candidate substance-untreated group.

In one embodiment of the present invention, the cell may be a liver cell.

In another embodiment of the present invention, the candidate substance may be selected from the group consisting of a compound, a microbial culture or extract, a natural product extract, a nucleic acid, and a peptide.

In another embodiment of the present invention, the nucleic acid comprises siRNA, shRNA, microRNA, antisense RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino Lt; / RTI >

In yet another embodiment of the present invention, the step (b) may be performed by PCR, microarray, northern blotting, western blotting, enzyme immunoassay (ELISA ), Immunoprecipitation, immunohistochemistry, and immunofluorescence. In the present invention, the immunoassay method can be used to measure the amount of the antibody to be detected.

The present inventors have found that secretion of IL-32 is induced by TNF-a and IFN-y cytokines secreted from infected cells when host cells are infected with HBV, secreted IL-32 increases phosphorylation of ERK1 / 2 The expression of HNF4α and HNF1α, which are the transcription factors of the host, involved in the transcription of the viral gene is reduced, thereby exhibiting DNA replication and gene expression inhibitory effects of the virus. When IL-32 is expressed in cells, The results showed that the antiviral effect of IL-32 mediated hepatitis B virus and its molecular mechanism were first identified. Therefore, the effect on IL-32 mediated HBV will provide a new understanding for the development of a therapeutic agent for the virus, and the composition for antiviral according to the present invention may be useful for the development of an antiviral therapeutic agent.

FIGS. 1A and 1B show the induction of IL-32 expression by TNF-α and IFN-γ. FIG. 1A shows the expression of TNF-α and IFN-γ in Huh7 hepatoma cell line by Western blotting The results of measurement of IL-32 protein expression level and changes of cell viability by the two types of cytokine treatment were measured. FIG. 1B shows that IL-32 protein is mainly expressed in the cytoplasm through a microscopic observation.
FIGS. 2A to 2D are results of confirming the effect of inhibiting replication of HBV by IL-32, wherein FIG. 2A shows no effect of inhibiting HBV replication by treatment of recombinant IL-32 (rhIL-32γ) protein in both Huh7 and HepG2 cells FIG. 2B shows that IL-32γ is mostly present in the cells through ELISA. FIG. 2C shows that the inhibition of replication of HBV is induced by treatment with TNF-α and IFN-γ in Huh7 cells FIG. 2d shows that the effect of inhibiting HBV replication is not shown by IL-32-specific siRNA treatment.
3A to 3D show the results of confirming the HBV replication inhibitory effect by the ectopic expression of IL-32. FIG. 3A shows the results of transfection of HBV 1.2 and IL-32 gamma expression plasmids into Huh7 cells, FIG. 3B shows the results of confirming the antiviral effect of IL-32 expressed in the cells by treating the IL-32 antibody with the culture medium, and FIG. 3C shows the results of the anti- 32 siRNA was transfected after expression of IL-32 gamma, and HBV transfection was inhibited by HBV < RTI ID = 0.0 > This is the result that shows no replication suppression.
4A to 4D are graphs showing inhibition of viral gene expression by IL-32 HBV enhancer down-regulation. FIG. 4A shows the results of transfection of Huh7 cells with HBV 1.2 and IL-32γ expression plasmids and Northern blot analysis of HBV mRNA FIG. 4B shows that the surface and core protein expression levels of HBV are decreased dependently on the IL-32γ transfection concentration. FIG. 4C shows the expression of HBV mRNA expression by IL-32γ And HBV enhancer luciferase reporter plasmid variants prepared for the present invention, and Fig. 4D is a graph showing the decrease in HBV enhancer activity by IL-32? As a result of Luciferase reporter analysis.
FIGS. 5A to 5F show the results of confirming the down-regulation of HNF4α and HNF1α transcription factors by IL-32. FIG. 5A shows binding sites of host transcription factors involved in HBV RNA transcription on the HBV enhancer gene map, And Fig. 5C are results showing that RNA and protein expression levels of HNF4 [alpha] and HNF1 [alpha] are decreased when Huh7 cells express IL-32γ through quantitative RT-PCR and Western blot respectively, FIG. 5f shows that the binding efficiency of HNF1α bound to RN (Enhancer I) and RII (Enhancer II) bound to RNI (Enhancer II) decreased under the condition that IL-32γ was expressed through IL- In the presence of HNF4α, the binding efficiency of the HNF4α-binding virus enhancer was decreased. At the same time, Western blotting using a native gel was carried out to confirm the expression of HNF4α protein at the corresponding site .
6a to 6e show that down-regulation of HNF4? And HNF1? By IL-32 is due to the ERK1 / 2-dependent pathway. FIG. 6a shows that phosphorylated ERK1 / 2 and p38 FIG. 6B shows that the expression of HNF4α and HNF1α decreased by IL-32 when treated with the ERK1 / 2 inhibitor U0126, and FIG. 6C and FIG. 6D show the results FIG. 6E shows that HBV replication level is increased again when U0126, an ERK1 / 2 inhibitor, is treated. FIG. 6E shows that HBV replication is increased again when HNF4α and HNF1α are overexpressed.
FIGS. 7A to 7C show results of confirming the HBV antiviral effect by IL-32 in a mouse model. FIG. 7A is a graph showing the results of immunization with mice expressing IL-32γ by injecting HBV 1.2 and IL-32γ expression plasmids into mice and Southern blotting FIG. 7B is a graph showing a decrease in HBsAg level due to the expression of IL-32γ in mouse serum. FIG. 7C shows the result of immunohistochemical staining using mouse liver tissue, showing that IL-32γ The expression of HBV surface protein was markedly decreased.
FIG. 8 is a graph showing the antiviral effect and the molecular mechanism of HBV induced by IL-32 identified in the present invention.

The present invention relates to an antiviral composition for hepatitis B virus comprising, as an active ingredient, interleukin-32 (IL-32), an expression or activity inhibitor of HNF4α (Hepatocyte nuclear factor 1 alpha) or HNF1α And a method for screening an antiviral substance against the virus.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by identifying the factors that induce antiviral effects by TNF-α and IFN-γ and their molecular mechanisms of action in host immune defense systems against hepatitis B virus.

Accordingly, the present invention relates to a method for inhibiting the expression or activity of HNF4α (Hepatocyte nuclear factor 1 alpha) or HNF1α (Hepatocyte nuclear factor 1 alpha), which comprises an interleukin-32 (IL-32) gene or an interleukin- There is provided an antiviral composition for hepatitis virus.

The term " antivirus " used in the present invention means to inhibit the proliferation of viruses in the body to weaken or extinguish the action of viruses invading into the body. More specifically, Or virus replication process, thereby inhibiting the proliferation of virus. In the present invention, hepatitis B virus is targeted.

In the present invention, HNF4α and HNF1α are nuclear transcription factors that bind to DNA, and HNF4α regulates the expression of various genes including HNF1α and regulates the expression of various genes in hepatocytes. It is also known to play an important role in liver, kidney, and intestinal development and clinically associated with diabetes and colon cancer, called maturity onset diabetes of the young (MODY). It has also been reported that it binds to the enhancer of the virus and promotes the transcription of the viral gene.

The term " activity inhibitor " used in the present invention means that the function of the target protein is degraded, and thus the function of the target protein is undetectable or present at a meaningless level.

The IL-32 gene, which is an expression or activity inhibitor of HNF4? And HNF1? Of the present invention, may be composed of a base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and more specifically, SEQ ID NO: 32?, IL-32?, IL-32?, And IL-32?, Preferably the IL-32 gene comprises the nucleotide sequence of SEQ ID NO:

In the present invention, the IL-32 protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NOS: 5 to 8, more specifically, SEQ ID NOS: 5 to 8 are IL-32 alpha, IL -32 [beta], IL-32 [gamma], and IL-32 delta protein, preferably the IL-32 protein comprises the amino acid sequence of SEQ ID NO:

Hereinafter, the antiviral effect and the molecular mechanism of IL-32-mediated hepatitis B virus infection were examined through the examples of the present invention.

In one embodiment of the present invention, in order to determine whether IL-32 expression is induced by cytokines TNF-a and IFN-y secreted from immune cells in viral infection, TNF-a and IFN- As a result, the expression of IL-32 was increased and IL-32 was more strongly induced when the two kinds of cytokines were treated together (see Example 2).

In another embodiment of the present invention, it was confirmed that HBV replication was inhibited by IL-32 induced by TNF-α and IFN-γ in hepatocarcinoma cell line, and it was confirmed that when the recombinant IL-32 protein was treated extracellularly (See Example 3).

In another embodiment of the present invention, HBV replication inhibitory effect is shown when inducing intracellular expression of IL-32 by transfecting plasmids expressing HBV and IL-32 in liver cancer cell line. IL-32 It was confirmed that HBV replication was inhibited by only IL-32 expressed in cells through the antiviral effect when the specific antibody was treated. In addition, all of the various isoform proteins of IL-32 showed the ability to inhibit HBV replication, and the effect of IFN-y was the best among them (see Example 4).

In another embodiment of the present invention, it has been confirmed that the above-described effect of inhibiting HBV replication by IL-32 appears at the level of RNA transcription of HBV, which is confirmed to be caused by a decrease in HBV enhancer activity (see Example 5) .

In another embodiment of the present invention, RNA and protein expression of HNF4 [alpha] and HNF1 [alpha] is reduced under conditions in which IL-32 is expressed by measuring the expression levels of transcription factors present in host hepatocytes involved in transcription by binding to the HBV enhancer , And the degree of binding of HNF4? And HNF1? To the HBV enhancer was decreased. Thus, the expression and activity of HNF4α and HNF1α were inhibited by IL-32, and transcription of HBV gene and its replication were inhibited (see Example 6).

In another embodiment of the present invention, the level of protein expression of molecules involved in MAPK signaling and antiviral signal transduction pathways associated with TNF-a and IFN-y under IL-32 expression conditions was measured and the results of phosphorylated ERK1 / 2 and p38 expression was decreased, and the expression level of HNF4α and HNF1α decreased by the expression of IL-32 upon the treatment with ERK1 / 2 inhibitor increased again, and the level of HBV replication again increased (see Example 7).

In another embodiment of the present invention, an antiviral effect by IL-32 expression was confirmed using an HBV-infected mouse model (see Example 8).

In the present invention, the expression of IL-32 is induced by the TNF-α and IFN-γ secreted from the infected cells during HBV infection, thereby increasing the phosphorylation of ERK1 / 2, HNF4 < / RTI > and < RTI ID = 0.0 > HNF1a < / RTI > involved in the expression of HBV and inhibit HBV gene expression and its replication by decreasing HBV enhancer activity.

The antiviral composition of the present invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The antiviral composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, subcutaneously, subcutaneously, nasally, or airway) according to a desired method, The severity of the disease, the form of the drug, the route of administration and the time, but may be suitably selected by those skilled in the art.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.001 to 150 mg, preferably 0.01 to 100 mg, One to three doses may be administered. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The composition of the present invention can be used in various forms such as pharmaceuticals, foods and beverages, and can be used in the form of powders, granules, tablets, capsules or drinks.

In another aspect of the present invention, the present invention provides a method for screening an antiviral substance against hepatitis B, comprising the following steps.

(a) treating the candidate material in cells in vitro ;

(b) measuring the level of IL-32 expression in said cells; And

(c) selecting an antiviral substance against hepatitis B as a substance that increases the expression of IL-32 as compared to a candidate substance-untreated group.

In the present invention, the cells include hepatocytes, and any kind of cells derived from the liver is not limited.

The candidate substance may be selected from the group consisting of a compound, a microbial culture or an extract, a natural product extract, a nucleic acid, and a peptide, and the nucleic acid preferably includes siRNA, shRNA, microRNA, antisense RNA, aptamer, LNA a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid, a nucleic acid,

The method for measuring the expression level of interleukin-32 in the step (b) includes a PCR, a microarray, a northern blotting, a western blotting, an enzyme But are not limited to, methods selected from the group consisting of immunoassay (ELISA), immunoprecipitation, immunohistochemistry, and immunofluorescence.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental Preparation and Experimental Method

1-1. Cell culture

Human liver cancer cell lines, Huh7 and HepG2, were purchased from the American Type Culture Collection (Manassas, Va., USA) and incubated with 10% fetal bovine serum (Gibco BRL) and 1% penicillin and streptomycin (Gibco BRL) The cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco BRL, Oregon, USA) medium at 37 ° C and 5% CO 2 .

1-2. Plasmid preparation

The HBV 1.2 plasmid used in the examples of the present invention was purchased from a DNA prep kit (Alphagene, Gyeonggi, Korea) and the pCAGGS mock vector, IL-32α, IL-32β and IL-32γ plasmids were supplied from other teams of Konkuk University Respectively. The HNF4a expression plasmid was amplified by PCR using the HepG2 cDNA library as template and subcloned into the pcDNA3.1 (+) vector (Invitrogen, Carlsbad, Calif.). The HBV enhancer luciferase plasmid was amplified by PCR using HBV 1.2 plasmid. In order to prepare the HBV enhancer luciferase reporter, the HBV 1.2 plasmid was used for amplification by PCR, and the primer sequences used at this time were as shown in Table 1 below.

primer order SEQ ID NO: pEnhI · II-Luc _Forward 5'-GGG GTA CCT AAA TAG ACC TAT TGA T-3 ' 9 pXp · EnhⅡ-Luc _Forward 5'-GGG GTA CCG CGC ATG C-3 ' 10 pNRE · EnhⅡ-Luc
_Forward 5'-GGG GTA CCG GAA ATA CAC CTC-3 ' 11 pEnhII / cp-Luc _Forward 5'-GGG GTA CCT CAC CTC TGC-3 ' 12 pEnhI · ΔII-Luc_Reverse 5'-AGA GAT CTA GCG AAG TCA CAC-3 ' 13 pEnh-Luc_Reverse 5'-TGA GAT CTA CAG ACC AAT TTA TGC-3 ' 14

1-3. Transfection and treatment conditions

Transfection was performed according to the manufacturer's protocol using Lipofectamine 2000 (Invitrogen) when the cells reached 70-80% of the culture vessel area. (YbdY, Seoul, Korea), TNF-α (YbdY), IFN-γ (LG, Jeonbuk, Korea) and anti-IL-32 And then cultured for 3 days. The cytokine treatment concentrations were determined using rhIL32 (0.1, 0.25, and 0.5 μg / ml), TNF-α (0.02 and 0.1 μg / ml), IFN- 0.02 / / ml and IFN-γ 500 U / ml). U0126, a signal transduction inhibitor, was added to a final concentration of 10 μM, and anti-IL32 was treated at a concentration of 0.2 and 0.5 μg ml -1. Transfected cells were recovered 2-3 days after transfection and the following experiments were carried out.

1-4. The RNA interference of IL-32 (RNA interference)

In order to specifically inhibit the expression of IL-32 mRNA, IL-32 specific siRNA was synthesized in ST Pharm (Seoul, Korea) and used for the experiments. The sequence of the siRNA is shown in Table 2 below and transfected into cells using Lipofectamine 2000 at a concentration of 10 nM or 20 nM.

siRNA order SEQ ID NO: IL-32 siRNA sense 5'-GGC UUA UGA UGA GGA GCA GTT-3 ' 15 antisense 5'-CUG CUC CUC AUA AUA AGC CTT -3 ' 16

1-5. Cell viability assay

Cell viability kits were purchased from WELGENE (Seoul, Korea) for analysis of cell viability. Huh7 cells were plated in 6-well plates and transfected or otherwise processed, the cell culture medium was replaced, and the original medium was transferred to a 96-well plate. Then, the XTT reagent and the PMS reagent were added to the 96-well plate and cultured for 1 hour, and the absorbance was measured at 450 nm using a spectrophotometer.

1-6. Southern blot

After 3 days, the cells were recovered with a scraper and 100 μl of HEPES buffer was added to dissolve the cells. Core capsids of HBV were precipitated with a polyethylene glycol (PEG) solution . Next, the core capsid was treated with SDS solution containing Proteinase K, and allowed to react at 37 ° C for 3 hours for decomposition. Total DNA was then separated from 1% agarose gel for 3 hours at 100 V and transferred to a nitrocellulose membrane (GE healthcare). To detect HBV DNA on the membrane, the membrane was hybridized with a random hexamer and a purified 32P labeled HBV probe.

1-7. Western blot

Cells were transfected and 2 or 3 days later, the cells were recovered and the cells were washed with RIPA buffer [20 mM Tris / HCl, 1% NP-40, 0.5% protease inhibitor cocktail (Sigma, St. Louis, NaCl, 2 mM KCl, pH 7.4]. After filtering the cell precipitate, the protein in the cell lysate was subjected to SDS-PAGE and classified by size. Next, the proteins in the polyacrylamide gel were transferred onto the PVDF membrane through a transfer process, and then the primary antibody diluted 1: 2000, that is, anti-IL-32γ (YbdY), anti-actin (Sigma) anti-HNF1? (Santa Cruz), anti-CEBP? (Santa Cruz), anti-HNF4? (Santa Cruz), anti-GFP (Sigma), anti-HBsAg (Santa Cruz), anti-lamin (Santa Cruz), or anti-tubulin (Santa Cruz). Then, the secondary antibody against the primary antibody was treated to confirm the expression level of the target protein. The membrane was treated with the stripping reagent to remove all of the reacted antibodies, and then used again to confirm the expression levels of the other proteins.

1-8. Luciferase reporter assay < RTI ID = 0.0 >

To measure the HBV enhancer activity, Luciferase reporter assays were performed according to the following method. More specifically, Huh7 cells were plated in 12-well culture plates at 2 × 10 5 cells / well and 0.5 μg of Enhancer-Luc (pEnhI.II, pEnhI.del II, pXp.EnhII, pEnhII / cp) 32 [gamma], and 0.25 [mu] g of [beta] -gal plasmid were transfected. At this time, pCAGGS was used as a control vector in order to standardize the amount of plasmid DNA that was total transfected. Forty-eight hours after transfection, cells were harvested and lysed and luciferase activity was measured using the reagent Steady Glo-Luciferase system (Promega, Madison, Wis.). Experimental data were repeated at least three times to derive the results.

1-9. Real-time quantitative PCR

Huh7 cells were lysed using the trizol reagent (Invitrogen) according to the manufacturer's protocol and total RNA was extracted. Using 2 μg of the extracted RNA and MMLV reverse transcriptase (Intron Biotechnology), a reaction solution was prepared so that the final mixed solution was 20 μl, followed by reverse transcription (RT-PCR) to synthesize cDNA. The cDNA synthesized according to the above procedure was subjected to a first denaturation at 94 ° C for 5 minutes, followed by a cycle of 94 ° C (30 seconds) and 72 ° C (1 minute) for 40 cycles, and a final extension at 72 ° C for 5 minutes Lt; / RTI > The primer sequences used are shown in Table 3 below. Real-time quantitative PCR amplification was performed using the ABI PRISM 7500 sequence detection machine using the SYBR Green PCR master mix (Applied Biosystems). The relative mRNA quantitation was performed using the ΔΔCt method and the result was calibrator (RQ = 2 -ΔΔCt ) Fold relative to the n-fold difference.

primer order SEQ ID NO: HNF1? Forward 5'-TGTGCGCTATGGACAGCCTGC-3 ' 17 Reverse 5'-CTGTGTTGGTGAACGTAGGA-3 18 HNF4? Forward 5'-GAGTGGGCCAAGTACATCCCAG-3 ' 19 Reverse 5'-GCTTTGAGGTAGGCATACT-3 ' 20 HNF3b Forward 5'-AAGATGGAAGGGCACGAGC-3 ' 21 Reverse 5'-TGTACGTGTTCATGCCGTTCA-3 ' 22 C / EBPa Forward 5'-CCTTGTGCAATGTGAATGTGC-3 ' 23 Reverse 5'-CGGAGAGTCTCATTTTGGCAA-3 ' 24 GAPDH Forward 5'-ATCATCCCTGCCTCTACTGG-3 ' 25 Reverse 5'-TGGGTGTCGCTGTTGAAGTC-3 ' 26

1-10. Chromatin immunoprecipitation assay (ChIP)

ChIP analysis was performed using the ChIP assay kit (Millipore, Billerica, MA) according to the following method. The supernatant was purified using protein A-agarose and incubated with anti-HNF1α (Santa Cruz), anti-HNF4α (Santa Cruz), or negative rabbit IgG antibody treated with negative control. Huh7 cells were cultured in 6-well plates and used for experiments after or without IL-32γ transfection. The primer sequences used in this experiment are shown in Table 4 below.

primer order SEQ ID NO: ChIP R1 Forward 5 '-TAAATAGACCTATTGATTGGAAAGTATGT-3' 27 Reverse 5 '-GAGAGAGGACAACAGAGTTGTCAG-3' 28 ChIP R2 Forward 5 '-TCACCTCTGCACGTCGCATG-3' 29 Reverse 5 '-ACAGACCAATTTATGCCTACAGCC-3' 30 ChIP R3 Forward 5 '-CTACTGTACCTGTCTTTAATCCTGAGTGG-3' 31 Reverse 5 '-CTGTGTGTAGTTTCTCTCTTATATAGAATG-3' 32

1-11. Electrophoretic mobility shift assay (EMSA)

Huh7 cells were transfected and 18 hours later, the cells were harvested and separated into nuclear and cytoplasmic fractions using Nuclear and Cytoplasmic Extraction Reagents (Thermo, Rockford, USA). Next, 2 ug nuclear fractions were preincubated and [32P] -gamma-labeled dsDNA oligonucleotides were used for HNF4α binding. The double-stranded DNA (Enhancer I HNF4?) Was purchased from bioneer (Daejeon, Korea). After the binding reaction was carried out on ice, the DNA-protein complex was subjected to electrophoresis at a low temperature using 6% polyacrylamide gel, and the gel was dried at 70 ° C for 30 minutes. Unlabeled HNF4α DNA was added and incubated for 10 min before [32P] -gamma-labeled probe treatment.

1-12. Immunofluorescence analysis

Immunofluorescence analysis was performed to observe the expression of IL-32γ, HNF4α, and HNF1α. Huh7 cells were transfected with IL-32γ plasmid and treated with TNF-α and IFN-γ, and after 48 hours, cells were observed with a confocal microscope (FV-1000 spectral, Olympus). More specifically, Huh7 cells were cultured to 50% of the cover glass area, and after 48 hours of transfection, the cells were fixed with acetone by treatment. Then, the cells were treated with 3% BSA solution diluted with PBS, followed by blocking reaction at 4 ° C for 18 hours at a low temperature, followed by washing with PBS. The antibody diluted at a ratio of 1: 300 was then treated at 4 ° C Lt; / RTI > overnight. The cells were again washed with PBS and the cells were treated with Alexa 488 and 568 secondary antibodies (goat and rabbit), reacted at room temperature for 1 hour, and washed with PBS. Toppro-3 (1: 500) was used to stain DNA in the nucleus of the cells. The coverslips were then mounted on a slide glass and mounted, and the cells were observed under a confocal microscope.

1-13. Hydrodynamic injection

Plasmid DNA (HBV 1.2, IL-32γ, and β-gal) was injected into male six-week-old male BALB / C mice by hydrodynamic injection. Plasmid DNA was added to PBS through the tail vein of the mouse, DNA was injected into the vein by high pressure for 4 to 6 seconds. All experiments were conducted with the approval of the Experimental Animal Care Committee of Konkuk University.

1-14. Immunohistochemical staining of mouse liver tissue

To detect IL-32γ in HBV-infected liver tissue, immunohistochemical staining was performed using mouse liver tissue infected with HBV by hydrodynamic injection. The mice were infected with HBV and after 4 days, the mice were sacrificed, the liver was extracted, and some liver tissues were fixed and embedded in a paraffin block. Then, the paraffin block was cut to a thickness of 5 μm using silane coated glasses (MUTO, Japan). Next, the sectioned slices were washed with alcohol, pretreated with 0.01 M sodium citrate (pH 6.0), diluted to a concentration of 3% in methanol to inactivate intracellular peroxidase Hydrogen peroxide (H 2 O 2 ) was treated. To prevent nonspecific binding, blocking sections were then applied to the tissue section samples, treated with IL-32 antibody, and reacted overnight at room temperature. After the reaction, 3,3'-diaminobenzidine tetrahydrochloride chromogen solution (DAKO) was applied to the tissue section slides, treated with hematoxylin for contrast dyeing and mounted with mounting solution.

Example 2. IL-32 induction in TNF-a and IFN-y mediated cells

Virus-specific CD8 + cytotoxic T cells can secrete TNF-α and IFN-γ. Cytokines secreted from these immune cells enable the host to protect against viral infections. Thus, in order to confirm the induction of IL-32 expression in cells by TNF-α and IFN-γ, Huh7 cells were treated with TNF-α and IFN-γ, respectively, Followed by Western blotting to measure the expression level of IL-32 protein.

As a result, as shown in FIG. 1A, expression of IL-32 was increased in proportion to the treatment concentration of TNF-α and IFN-γ in the cells. When two kinds of cytokines were treated together, IL-32 protein Secretion was strongly induced. At this time, in order to examine whether TNF-? And IFN-? Treatment had an effect on cell viability, the cytokines were treated with the respective cytokines for 48 hours, and XTT assay was performed to confirm that there was no change in cell viability. In addition, as shown in FIG. 1B, it was confirmed that the IL-32 protein was mainly expressed in the cytoplasm through microscopic observation together with the Western blot result.

3. Inhibition of HBV replication by IL-32 induced by TNF-α and IFN-γ

The results of Example 2 above confirmed that IL-32 expression was induced in cells by TNF-α and IFN-γ, and furthermore, to test whether IL-32 exhibits an anti-HBV effect. Huh7 and HepG2 cells were treated with recombinant human IL-32γ (rhIL-32γ) at concentrations of 0.1, 0.25, and 0.5 μg, respectively.

As a result, as shown in Fig. 2A, it was confirmed that no effect of inhibiting HBV replication was observed by rhIL-32? In both types of cells. To investigate the biological activity of rhIL-32γ on the basis of the above results, human THP-1 and mouse Raw 264.7 cells were treated with two different concentrations of rhIL-32γ, the most active form of IL-32, -32 was measured. As shown in FIG. 2B, it was confirmed that IL-32 gamma was more abundant in the cells than in the culture solution. In addition, IL-32 was expressed in most of the cells even when TNF-a and IFN-y were treated, respectively.

Based on the above results, we evaluated the anti-HBV effect by TNF-α and IFN-γ treatment in Huh7 cell line. For this, the cells were treated with TNF-α (20 ng or 100 ng) or IFN-γ (500 U or 1000 U), respectively, or with 20 ng of TNF-α and IFN-γ 500 U Southern blotting was carried out according to the method of Example 1-6. As a result, as shown in FIG. 2C, the antiviral effect against HBV was shown by treatment with TNF-α and IFN-γ, and when the two types of cytokines were treated together, the antiviral effect was strongly induced. Furthermore, it was confirmed that there was no change in cell viability due to the treatment of the above substances.

To further confirm the antiviral effect, the antiviral effect on HBV was measured after inhibiting the expression of IL-32 by transfecting Huh7 and HepG2 cell lines with IL-32 specific siRNA. As a result, as shown in Fig. 2 (d), it was confirmed that HBV replication which was inhibited by inhibition of IL-32 expression was restored again.

These results indicate that IL-32 acts as a sub-marker molecule of TNF- [alpha] and IFN- [gamma], and its intracellular expression mediates the antiviral effect on HBV.

Example 4 Confirmation of HBV Repression Inhibition by Expression of IL-32

In order to examine the possible regulatory effect of IL-32γ on HBV replication, HBV 1.2 and IL-32γ plasmids were transfected into Huh7 and HepG2 cells, and HBV DNA was extracted and subjected to Southern blotting to obtain HBV The degree of replication was analyzed.

As a result, as shown in FIG. 3A, the level of HBV DNA was significantly decreased depending on the expression level of IL-32γ, and the phosphoimager result showed that DNA replication of HBV was reduced by about 70 to 80% -32 expression level of the cells.

In addition to the above results, in order to examine whether IL-32 secreted out of the cell affects the ability to inhibit HBV replication in the cells, IL-32 antibody was treated with Huh7 cell culture medium to induce neutralizing action on IL-32 in the culture medium The titer of HBV was measured. As a result, as shown in FIG. 3B, it was confirmed that even when the antibody against IL-32 was treated, there was no change in the degree of inhibition of HBV replication by IL-32. This result implies that replication of HBV is inhibited by IL-32? Expressed in cells only.

Furthermore, in order to examine whether other isoforms of IL-32 exhibit an antiviral effect such as IL-32γ, Huh7 cells were transfected with IL-32α, IL-32β, IL-32γ plasmids or TNF- α and IFN-γ, and then subjected to Southern blotting to compare the degree of HBV replication. As a result, as shown in FIG. 3C, both IL-32α, IL-32β and IL-32γ decreased HBV replication, and it was confirmed that the antiviral effect of IL-32γ was the most excellent. Further, as shown in FIG. 3D, in order to confirm the effect of inhibiting HBV replication by IL-32γ expression, it was confirmed that when the expression of IL-32 was inhibited by transfection of IL-32 siRNA, Respectively.

Example 5. Confirmation of Transcription of Viral Gene and Suppression of Protein Expression through Down-regulation of IL-32-mediated Enhancer Activity

Based on the results of Examples 3 and 4, in order to examine at which stage HBV replication was inhibited by intracellular IL-32 expression, experiments were carried out in the same manner as in Example 4, and HBV mRNA expression levels were measured. As a result, it was confirmed that the levels of HBV mRNA such as pg / preC RNA and HBV surface RNA (Pre-S / S RNA) were decreased depending on the transfection concentration of IL-32γ as shown in FIG. In addition, as shown in FIG. 4B, it was confirmed that the surface and core protein levels of HBV significantly decreased in proportion to the transfection concentration of IL-32γ.

Based on the above results, the HBV enhancer activity, which plays an important role in mRNA transcription of HBV, was measured by Luciferase reporter assay in order to investigate the mechanism by which IL-32γ reduces the mRNA level of HBV. For this, HBV wild-type enhancers as HBV enhancer luciferase reporter plasmids and mutants in which some of them were deleted were used as shown in Fig. 4C. As a result, as shown in Fig. 4D, it was confirmed that the HBV enhancer activity was reduced by IL-32?. This suggests that inhibition of the replication of HBV by IL-32γ in Huh7 cells is due to inhibition of the activity of the HBV enhancer.

Example 6. Downregulation of HNF4 < alpha > and HNF1 alpha binding to the enhancer of HBV by IL-32

Recent studies have reported that RNA production of HBV is associated with various transcription factors, HNF1, HNF3, HNF4, and CEBP, which are abundant in hepatocytes. This means that HBV does not produce transcription factors but uses transcription factors that are present in host cells in their replication process. Therefore, the inventors of the present invention carried out experiments in anticipation that transcription factors present in such hepatocytes would be related to the inhibition of HBV RNA transcription by IL-32.

First, the binding sites of various transcription factors related to HBV RNA transcription on the enhancer gene map of HBV were analyzed. As a result, as shown in FIG. 5A, the four types of CEBP, HNF1, HNF3, and HNF4 transcription factors can bind to different sites of the enhancer I / II sequence, There were close or overlapping areas. In addition, each transcription factor exhibits a unique function by binding to a specific site of the enhancer. For example, HNF4 promotes transcription of the virus, and HNF3 inhibits HBV transcription.

Thus, in order to find a transcription factor that reduces the enhancer activity of HBV virus, the expression levels of the transcription factors present in hepatocytes in Huh7 cells were quantitatively analyzed by quantitative RT-PCR and Western blotting according to the methods of Examples 1-9 and 1-7. Analysis. As a result, as shown in FIG. 5B and FIG. 5C, when the expression of IL-32γ was decreased, the expression of HNF4α and HNF1α RNA and protein were all decreased, and the expression levels of HNF3 and CEBP did not show any difference.

Furthermore, in order to determine whether there is a correlation between IL-32 and transcription factors, ChIP analysis was performed according to the method of Example 1-10, and the transcription factors . Immunoprecipitated HBV DNA fragments were detected by semi-quantitative RT-PCR using anti-HNF4α and anti-HNF1α antibodies. As a result, as shown in FIG. 5D and FIG. 5E, the binding efficiency of HNF1α binding to RN (Enhancer I) and HNF1α binding to RII (Enhancer II) decreased in the condition where IL-32 was expressed. This result implies that IL-32 influences the enhancer-binding site of HBV through any method, and the binding pattern of the transcription factors is changed.

To further confirm the above results, EMSA and Western blotting were performed according to the method of Example 1-11, and western blotting was performed under the same conditions as EMSA. As a result, as shown in Fig. 5F, it was observed that the HNF4? Protein was present at the same position of the protein-DNA. However, in the presence of IL-32, the expression of HNF4α protein did not bind to the HNF4α probe due to decreased expression, which was consistent with the ChIP result.

These results indicate that intracellular IL-32 inhibits HBV replication by altering the binding pattern of transcription factors.

Example 7. Confirmation of inhibition of IL-32 mediated HBV replication through downregulation of ERK1 / 2-dependent HNF4?

TNF-α and IFN-γ induce activation of mitogen-activated protein kinases (MAPKs), and MAPK signaling in host cells is known to play an important role in antiviral signal transduction networks.

Therefore, the present inventors measured the levels of protein expression of several molecules related to MAPK signaling and antiviral signal transduction pathway under the condition of expressing IL-32. At this time, a sample treated with only TNF-? And IFN-? Was used as a positive control. As shown in FIG. 6A, when IL-32 was expressed in Huh7 cells, expression of phosphorylated ERK1 / 2 (pextracellular signal-regulated kinase 1/2) and p38 protein was increased, and JNK kinase) expression did not change.

Based on the above results, in order to examine how HNF4α and HNF1α exhibit such regulatory effect, U0126, an ERK1 / 2 inhibitor, was treated with 10 μM of cells to inhibit the production of p-ERK1 / 2. As a result, as shown in FIG. 6B, it was confirmed that the protein expression of HNF4α and HNF1α, which had been reduced by IL-32, was increased again when p-ERK1 / 2 protein was decreased. Furthermore, it is known that the expression of transcription factors present in the liver is regulated by the MAPK signaling pathway, and thus the expression of HNF4α and HNF1α is reduced by IL-32, which is mediated by the MAPK signaling pathway .

In addition, when the HNF4α and HNF1α were overexpressed, the replication level of the HBV virus was measured. As shown in FIGS. 6C and 6D, it was confirmed that replication of the virus decreased by IL-32 again. In addition, as can be seen in FIG. 6E, it was confirmed that the level of HBV replication decreased again by IL-32 when ERK1 / 2 phosphorylation inhibitor was also treated.

These results indicate that IL-32 inhibits HBV replication by decreasing expression of HNF4? And HNF1? Through induction of phosphorylation of ERK1 / 2.

Example 8. Confirmation of HBV Inhibition by IL-32 in Mouse Model

In order to verify the results derived from the above examples, we have examined the antiviral effect of IL-32 in a mouse model of HBV infection in vivo .

For this, HBV 1.2 expression plasmids and IL-32 gamma expression plasmids were injected into mice using the hydrodynamic injection technique. Then, HBV DNA was extracted from 50 ㎎ of mouse liver tissue and DNA replication level of HBV was measured through Southern blotting. As a result, as shown in FIG. 7A, it was confirmed that the HBV and IL-32 gamma expression plasmids were simultaneously injected into the mouse, and the HBV DNA replication level was decreased compared with the mice injected with the HBV expression plasmid alone. In addition, the same results were confirmed by the relative quantification of HBV DNA replication using phospho-imager.

In addition, as shown in FIG. 7B, the HBsAg level was significantly decreased upon expression of IL-32γ in mouse serum. The degree of expression of IL-32 and HBV surface proteins was examined by immunohistochemical staining. As a result, As shown, IL-32γ mediates the antiviral effect on HBV in mice through the marked reduction of HBV surface protein expression when IL-32γ is expressed.

The results are summarized in FIG. 8. More specifically, when HBV is infected into cells, HBV produces its own mRNA using HNF4α and HNF1α, the host's transcription factors. Immunocytes surrounding the infected cells secrete TNF-α and IFN- secrete < / RTI > These cytokines induce the secretion of IL-32 and the secreted IL-32 increases the phosphorylation of ERK1 / 2 thereby reducing the expression of the transcription factors HNF4a and HNF1a. As a result, in the present invention, it was confirmed that HBV DNA replication and viral RNA production are reduced by this mechanism of IL-32.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Konkuk University Glocal Industry-Academic Collaboration Foundation <120> Antiviral compositions against hepatitis B virus          interleukin-32 as an active ingredient <130> MP16-467 <160> 32 <170> KoPatentin 3.0 <210> 1 <211> 919 <212> DNA <213> IL-32α <400> 1 ccccagccag ctgtcccgag tctggacttt ccctctgccc ctccccactc tcaggctggt 60 ggggtgggga aagcagccca ttcctgggct cagagactcc caccccagct cagagggagc 120 aggggcccag ccagggacgg accctcattc ctcccaggga ccccagacct ctgtctctct 180 cgggccttgg ctccttgaac ttttggccgc catgtgcttc ccgaaggtcc tctctgatga 240 catgaagaag ctgaaggccc gaatgcacca ggccatagaa agattttatg ataaaatgca 300 aaatgcagaa tcaggacgtg gacaggtgat gtcgagcctg gcagagctgg aggacgactt 360 caaagagggc tacctggaga cagtggcggc ttattatgag gagcagcacc cagagctcac 420 tcctctactt gaaaaagaaa gagatggatt acggtgccga ggcaacagat cccctgtccc 480 ggatgttgag gatcccgcaa ccgaggagcc tggggagagc ttttgtgaca agtcctacgg 540 agccccacgg ggggacaagg aggagctgac accccagaag tgctctgaac cccaatcctc 600 cccggacccc tccctcagct gtcctgtgcc ccgccctctc ccgcacactc agtccccctg cctggcgttc 720 ctgccgcagc tctgacctgg tgctgtcgcc ctggcatctt aataaaacct gcttatactt 780 ccctggcagg ggagatacca tgatcgcgga ggtgggtttc ccagggcaag gctgatctgt 840 tgccgtatta gtccgttttc acacagctat aaagaatgcc tgagactggg tgatgtataa 900 agaaaagaag tttaactga 919 <210> 2 <211> 1157 <212> DNA <213> IL-32β <400> 2 ccccagccag ctgtcccgag tctggacttt ccctctgccc ctccccactc tcaggctggt 60 ggggtgggga aagcagccca ttcctgggct cagagactcc caccccagct cagagggagc 120 aggggcccag ccagggacgg accctcattc ctcccaggga ccccagacct ctgtctctct 180 cggctgagta tttgtgccag gaagactgcg tgcagaaggt gactgtctca gtggagctgg 240 gtcatctcag gccttggctc cttgaacttt tggccgccat gtgcttcccg aaggtcctct 300 ctgatgacat gaagaagctg aaggcccgaa tgcaccaggc catagaaaga ttttatgata 360 ggcggggg acgacttcaa agagggctac ctggagacag tggcggctta ttatgaggag cagcacccag 480 agctcactcc tctacttgaa aaagaaagag atggattacg gtgccgaggc aacagatccc 540 ctgtcccgga tgttgaggat cccgcaaccg aggagcctgg ggagagcttt tgtgacaagg 600 tcatgagatg gttccaggcc atgctgcagc ggctgcagac ctggtggcac ggggttctgg 660 cctgggtgaa ggagaaggtg gtggccctgg tccatgcagt gcaggccctc tggaaacagt 720 tccagagttt ctgctgctct ctgtcagagc tcttcatgtc ctctttccag tcctacggag 780 ccccacgggg ggacaaggag gagctgacac cccagaagtg ctctgaaccc caatcctcaa 840 aatgaagata ctgacaccac ctttgccctc cccgtcaccg cgcacccacc ctgacccctc 900 cctcagctgt cctgtgcccc gccctctccc gcacactcag tccccctgcc tggcgttcct 960 gccgcagctc tgacctggtg ctgtcgccct ggcatcttaa taaaacctgc ttatacttcc 1020 ctggcagggg agataccatg atcgcggagg tgggtttccc agggcaaggc tgatctgttg 1080 ccgtattagt ccgttttcac acagctataa agaatgcctg agactgggtg atgtataaag 1140 aaaagaagtt taactga 1157 <210> 3 <211> 705 <212> DNA <213> IL-32γ <400> 3 ccccagccag ctgtcccgag tctggacttt ccctctgccc ctccccactc tcaggctggt 60 ggggtgggga aagcagccca ttcctgggct cagagactcc caccccagct cagagggagc 120 aggggcccag ccagggacgg accctcattc ctcccaggga ccccagacct ctgtctctct 180 cgggccttgg ctccttgaac ttttggccgc catgtgcttc ccgaaggtcc tctctgatga 240 catgaagaag ctgaaggccc gaatggtaat gctcctccct acttctgctc aggggttggg 300 ggcctgggtc tcagcgtgtg acactgagga cactgtggga cacctgggac cctggaggga 360 caaggatccg gccctttggt gccaactctg cctctcttca cagcaccagg ccatagaaag 420 attttatgat aaaatgcaaa atgcagaatc aggacgtgga caggtgatgt cgagcctggc 480 agagctggag gacgacttca aagagggcta cctggagaca gtggcggctt attatgagga 540 gcagcaccca gagctcactc ctctacttga aaaagaaaga gatggattac ggtgccgagg 600 caacagatcc cctgtcccgg atgttgagga tcccgcaacc gaggagcctg gggagagctt 660 ttgtgacaag gtcatgagat ggttccaggc catgctgcag cggctgcaga cctggtggca 720 cggggttctg gcctgggtga aggagaaggt ggtggccctg gtccatgcag tgcaggccct 780 ctggaaacag ttccagagtt tctgctgctc tctgtcagag ctcttcatgt cctctttcca 840 gtcctacgga gccccacggg gggacaagga ggagctgaca ccccagaagt gctctgaacc 900 ccaatcctca aaatgaagat actgacacca cctttgccct ccccgtcacc gcgcacccac 960 cctgacccct ccctcagctg tcctgtgccc cgccctctcc cgcacactca gtccccctgc 1020 ctggcgttcc tgccgcagct ctgacctggt gctgtcgccc tggcatctta ataaaacctg 1080 cttatacttc cctggcaggg gagataccct gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140 aaaaaaaaaa aaaaaaaaa 1159 <210> 4 <211> 880 <212> DNA <213> IL-32δ <400> 4 gccttggctc cttgaacttt tggccgccat gtgcttcccg aaggtcctct ctgatgacat 60 gaagaagctg aaggcccgaa tgcaccaggc catagaaaga ttttatgata aaatgcaaaa 120 tgcagaatca ggacgtggac aggacgactt caaagagggc tacctggaga cagtggcggc 180 ttattatgag gagcagcacc cagagctcac tcctctactt gaaaaagaaa gagatggatt 240 acggtgccga ggcaacagat cccctgtccc ggatgttgag gatcccgcaa ccgaggagcc 300 tggggagagc ttttgtgaca aggtcatgag atggttccag gccatgctgc agcggctgca 360 gacctggtgg cacggggttc tggcctgggt gaaggagaag gtggtggccc tggtccatgc 420 agtgcaggcc ctctggaaac agttccagag tttctgctgc tctctgtcag agctcttcat 480 gtcctctttc cagtcctacg gagccccacg gggggacaag gaggagctga caccccagaa 540 gtgctctgaa ccccaatcct caaaatgaag atactgacac cacctttgcc ctccccgtca 600 ccgcgcaccc accctgaccc ctccctcagc tgtcctgtgc cccgccctct cccgcacact 660 cagtccccct gcctggcgtt cctgccgcag ctctgacctg gtgctgtcgc cctggcatct 720 taataaaacc tgcttatact tccctggcag gggagatacc atgatcgcgg aggtgggttt 780 cccagggcaa ggctgatctg ttgccgtatt agtccgtttt cacacagcta taaagaatgc 840 ctgagactgg gtgatgtata aagaaaagaa gtttaactga 880 <210> 5 <211> 131 <212> PRT <213> IL-32α <400> 5 Met Cys Phe Pro Lys Val Leu Ser Asp Asp Met Lys Lys Leu Lys Ala   1 5 10 15 Arg Met His Gln Ala Ile Glu Arg Phe Tyr Asp Lys Met Gln Asn Ala              20 25 30 Glu Ser Gly Arg Gly Glu Val Met Ser Ser Leu Glu Leu Glu Asp          35 40 45 Asp Phe Lys Glu Gly Tyr Leu Glu Thr Val Ala Ala Tyr Tyr Glu Glu      50 55 60 Gln His Pro Glu Leu Thr Pro Leu Leu Glu Lys Glu Arg Asp Gly Leu  65 70 75 80 Arg Cys Arg Gly Asn Arg Ser Pro Val Pro Asp Val Glu Asp Pro Ala                  85 90 95 Thr Glu Glu Pro Gly Glu Ser Phe Cys Asp Lys Ser Tyr Gly Ala Pro             100 105 110 Arg Gly Asp Lys Glu Glu Leu Thr Pro Gln Lys Cys Ser Glu Pro Gln         115 120 125 Ser Ser Lys     130 <210> 6 <211> 188 <212> PRT <213> IL-32β <400> 6 Met Cys Phe Pro Lys Val Leu Ser Asp Asp Met Lys Lys Leu Lys Ala   1 5 10 15 Arg Met His Gln Ala Ile Glu Arg Phe Tyr Asp Lys Met Gln Asn Ala              20 25 30 Glu Ser Gly Arg Gly Glu Val Met Ser Ser Leu Glu Leu Glu Asp          35 40 45 Asp Phe Lys Glu Gly Tyr Leu Glu Thr Val Ala Ala Tyr Tyr Glu Glu      50 55 60 Gln His Pro Glu Leu Thr Pro Leu Leu Glu Lys Glu Arg Asp Gly Leu  65 70 75 80 Arg Cys Arg Gly Asn Arg Ser Pro Val Pro Asp Val Glu Asp Pro Ala                  85 90 95 Thr Glu Glu Pro Gly Glu Ser Phe Cys Asp Lys Val Met Arg Trp Phe             100 105 110 Gln Ala Met Leu Gln Arg Leu Gln Thr Trp Trp His Gly Val Leu Ala         115 120 125 Trp Val Lys Glu Lys Val Ala Leu Val His Ala Val Gln Ala Leu     130 135 140 Trp Lys Gln Phe Gln Ser Phe Cys Cys Ser Leu Ser Glu Leu Phe Met 145 150 155 160 Ser Ser Phe Gln Ser Tyr Gly Ala Pro Arg Gly Asp Lys Glu Glu Leu                 165 170 175 Thr Pro Gln Lys Cys Ser Glu Pro Gln Ser Ser Lys             180 185 <210> 7 <211> 234 <212> PRT <213> IL-32γ <400> 7 Met Cys Phe Pro Lys Val Leu Ser Asp Asp Met Lys Lys Leu Lys Ala   1 5 10 15 Arg Met Val Met Leu Leu Pro Thr Ser Ala Gln Gly Leu Gly Ala Trp              20 25 30 Val Ser Ala Cys Asp Thr Glu Asp Thr Val Gly His Leu Gly Pro Trp          35 40 45 Arg Asp Lys Asp Pro Ala Leu Trp Cys Gln Leu Cys Leu Ser Ser Gln      50 55 60 His Gln Ala Ile Glu Arg Phe Tyr Asp Lys Met Gln Asn Ala Glu Ser  65 70 75 80 Gly Arg Gly Gln Val Met Ser Ser Leu Ala Glu Leu Glu Asp Asp Phe                  85 90 95 Lys Glu Gly Tyr Leu Glu Thr Val Ala Ala Tyr Tyr Glu Glu Gln His             100 105 110 Pro Glu Leu Thr Pro Leu Leu Glu Lys Glu Arg Asp Gly Leu Arg Cys         115 120 125 Arg Gly Asn Arg Ser Pro Val Pro Asp Val Glu Asp Pro Ala Thr Glu     130 135 140 Glu Pro Gly Glu Ser Phe Cys Asp Lys Val Met Arg Trp Phe Gln Ala 145 150 155 160 Met Leu Gln Arg Leu Gln Thr Trp Trp His Gly Val Leu Ala Trp Val                 165 170 175 Lys Glu Lys Val Val Ala Leu Val His Ala Val Gln Ala Leu Trp Lys             180 185 190 Gln Phe Gln Ser Phe Cys Cys Ser Leu Ser Glu Leu Phe Met Ser Ser         195 200 205 Phe Gln Ser Tyr Gly Ala Pro Arg Gly Asp Lys Glu Glu Leu Thr Pro     210 215 220 Gln Lys Cys Ser Glu Pro Gln Ser Ser Lys 225 230 <210> 8 <211> 179 <212> PRT <213> IL-32δ <400> 8 Met Cys Phe Pro Lys Val Leu Ser Asp Asp Met Lys Lys Leu Lys Ala   1 5 10 15 Arg Met His Gln Ala Ile Glu Arg Phe Tyr Asp Lys Met Gln Asn Ala              20 25 30 Glu Ser Gly Arg Gly Gln Asp Asp Phe Lys Glu Gly Tyr Leu Glu Thr          35 40 45 Val Ala Ala Tyr Tyr Glu Glu Gln His Pro Glu Leu Thr Pro Leu Leu      50 55 60 Glu Lys Glu Arg Asp Gly Leu Arg Cys Arg Gly Asn Arg Ser Ser Pro Val  65 70 75 80 Pro Asp Val Glu Asp Pro Ala Thr Glu Glu Pro Gly Glu Ser Phe Cys                  85 90 95 Asp Lys Val Met Arg Trp Phe Gln Ala Met Leu Gln Arg Leu Gln Thr             100 105 110 Trp Trp His Gly Val Leu Ala Trp Val Lys Glu Lys Val Val Ala Leu         115 120 125 Val His Ala Val Gln Ala Leu Trp Lys Gln Phe Gln Ser Phe Cys Cys     130 135 140 Ser Leu Ser Glu Leu Phe Met Ser Ser Phe Gln Ser Tyr Gly Ala Pro 145 150 155 160 Arg Gly Asp Lys Glu Glu Leu Thr Pro Gln Lys Cys Ser Glu Pro Gln                 165 170 175 Ser Ser Lys             <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> pEnhI.II-Luc_Forward <400> 9 ggggtaccta aatagaccta ttgat 25 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> pXp.EnhII-Luc_Forward <400> 10 ggggtaccgc gcatgc 16 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pNRE.EnhII-Luc_Forward <400> 11 ggggtaccgg aaatacacct c 21 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> pEnhII / cp-Luc_Forward <400> 12 ggggtacctc acctctgc 18 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> pEnhI.ΔII-Luc_Reverse <400> 13 agagatctag cgaagtcaca c 21 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> pEnh-Luc_Reverse <400> 14 tgagatctac agaccaattt atgc 24 <210> 15 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> IL-32 siRNA_sense <400> 15 ggcuuauuau gaggagcagt t 21 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> IL-32 siRNA_antisense <400> 16 cugcuccuca uaauaagcct t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF1α_Forward <400> 17 tgtgcgctat ggacagcctg c 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HNF1α_Reverse <400> 18 ctgtgttggt gaacgtagga 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> HNF4α_Forward <400> 19 gagtgggcca agtacatccc ag 22 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF4α_Reverse <400> 20 gctttgaggt aggcatact 19 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HNF3b_Forward <400> 21 aagatggaag ggcacgagc 19 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HNF3b_Reverse <400> 22 tgtacgtgtt catgccgttc a 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C / EBPa_Forward <400> 23 ccttgtgcaa tgtgaatgtg c 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> C / EBPa_Reverse <400> 24 cggagagtct cattttggca a 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Forward <400> 25 atcatccctg cctctactgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_Reverse <400> 26 tgggtgtcgc tgttgaagtc 20 <210> 27 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> ChIP R1_Forward <400> 27 taaatagacc tattgattgg aaagtatgt 29 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ChIP R1_Reverse <400> 28 gagagaggac aacagagttg tcag 24 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ChIP R2_Forward <400> 29 tcacctctgc acgtcgcatg 20 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ChIP R2_Reverse <400> 30 acagaccaat ttatgcctac agcc 24 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> ChIP R3_Forward <400> 31 ctactgtacc tgtctttaat cctgagtgg 29 <210> 32 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ChIP R3_Reverse <400> 32 ctgtgtgtag tttctctctt atatagaatg 30

Claims (12)

delete delete delete delete delete A method of screening for an antiviral agent against hepatitis B comprising the steps of:
(a) treating a candidate substance in a liver cell in vitro ;
(b) measuring the expression level of interleukin-32 (IL-32) in the hepatocyte; And
(c) selecting an antiviral substance against hepatitis B as a substance that increases the expression of IL-32 as compared to a candidate substance-untreated group,
Wherein the hepatocytes are hepatocytes in which IFN-? 1 is not expressed.
delete The method according to claim 6,
Wherein the candidate substance is selected from the group consisting of a compound, a microorganism culture solution or extract, a natural product extract, a nucleic acid, and a peptide.
9. The method of claim 8,
Wherein the nucleic acid is selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino. Screening method.
The method according to claim 6,
The step (b) may be carried out by PCR, microarray, northern blotting, western blotting, enzyme immunoassay (ELISA), immunoprecipitation, An immunohistochemistry, and an immunofluorescence. The screening method according to claim 1,
delete delete
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CN110859840A (en) * 2019-11-29 2020-03-06 湖南大学 Application of nicotinic acid in preparing medicine for treating chronic hepatitis B
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