JP2005341864A - Jc virus granule formation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a peptide like compound inhibiting the granule formation of a JC virus in a living body, suppressing its propagation and also expected to contribute the development of treating method of human progressive multifocal leucoencephalopathy (PML), and a DNA encoding the same. <P>SOLUTION: It is elucidated that there are 2 sites of sequences performing an interaction with a VP1 which is a main outer shell protein of the JC virus and inhibiting the formation of the viral granules, in a peptide sequence originated from an agnoptotein of the JC virus. The peptide of the region having an effect on these physiological activity and the DNA encoding such peptide are made as the granule formation inhibitor of the JC virus and the preventing and treating agent of the PML. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a JC virus particle formation inhibitor comprising a peptide derived from JC virus Agnoprotein or a DNA encoding the same as an active ingredient, and prevention of human progressive multifocal leukoencephalopathy (PML). -It relates to therapeutic agents.

  Human progressive multifocal leukoencephalopathy (PML) is a demyelinating disease caused by infection with JC virus (JCV). JCV is a double-stranded circular DNA virus belonging to the genus Polyomavirus. Usually, 70% or more of the healthy human urinary system is subclinically infected, but when the host falls into an immunodeficient state, it moves to the brain. Infection with oligodendroglia causes fatal demyelination.

  Conventionally, PML has been a relatively rare disease, but has recently been increasing with the increase in AIDS patients and the introduction of bone marrow / organ transplantation treatment. In particular, in transplantation treatment, it is inevitable that a patient is exposed to an extremely immunosuppressed state, and it is considered that control of PML greatly affects the success or failure of transplantation treatment. Despite the current situation, the effects of cytosine arabinoside (Ara-C) and interferon β, which are DNA synthesis inhibitors that have been used in the treatment of PML, have not been clarified yet, and are not yet at this stage. An effective treatment has not been established.

As a result of researches aimed at elucidating the glial cell-specific infection mechanism of JC virus and establishing a therapeutic method for viral encephalopathy, the present inventors have previously analyzed Agnoprotein, an unknown protein encoded by JC virus. Has already shown that JCV promotes virus particle formation, and that JC viruses that have mutations in the agno gene and are unable to express Agnoprotein are significantly delayed in their growth, and genetic engineering techniques such as antisense oligos and RNAi It has been reported that artificially controlling the expression of agno gene by using or inhibiting the function of Agnoprotein using anti-Agnoprotein antibody can suppress the growth of JC virus and thus contribute to the development of therapeutic methods for PML. (For example, refer to Patent Document 1).
JP 2003-160510 A

  An object of the present invention is to provide peptide-like compounds that are expected to inhibit JCV particle formation in vivo, suppress their growth, and thus contribute to the development of therapeutic methods for PML, and DNA encoding the same. There is.

  Agnoprotein is encoded in the uppermost stream of the late protein transcription region of JCV, and a protein consisting of 71 amino acids (8 kDa) is transcribed and translated, but its function is unknown. It was found that there was a sequence that binds to VP1, which is the major outer protein of JCV, in the sequence, and the binding region between this Agnoprotein and VP1, which is the JCV outer shell protein, was determined. In addition, it is clear that the peptide sequence derived from Agnoprotein has one sequence that promotes particle formation (41st to 71st) and two sequences that inhibit (11th to 30th, and 48th to 71st). did. Peptides having the 11th to 30th and 41st to 71st sequences bind to the C-terminal GST fusion protein of VP1, so that Agnoprotein interacts with the C-terminal part of VP1 monomer or VP15-mer. It is presumed that it helps the formation of virus particles. Furthermore, it has been found that a peptide fragment having a partial amino acid sequence of Agnoprotein inhibits the formation of virus particles. That is, it has been found that new generation of JCV particles can be prevented by using a peptide-like compound having this sequence (virus particle formation inhibitory sequence). By coexisting a peptide having the 11th to 30th sequences with VP1, it is presumed that this auxiliary role was inhibited and the ability to form particles was reduced.

  A peptide having the 48th to 71st sequence is a GST fusion protein obtained by binding a fusion protein of glutathione-S-transferase (GST) with the N-terminal half or C-terminal half region of VP1 (each GST-VP1). (N) and GST-VP1 (C) were not bound to each other, but the particle forming ability was inhibited. This is presumed to be a mechanism in which this peptide recognizes and binds to the three-dimensional structure consisting of the full length of VP1 and inhibits the formation of particles. Viral particle maturation in the host cell is an important process in the new infection of other cells, along with the growth of the virus and the destruction of the host cell. According to the present invention, it has been suggested that the use of an Agnoprotein-derived peptide inhibits the formation of JCV particles, and as a result, the possibility of significantly suppressing virus growth. The present invention has been completed based on these findings.

That is, the present invention relates to (1) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and (2) an amino acid in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2. A peptide comprising a sequence and having a particle-forming ability inhibitory activity of JC virus, and (3) a DNA encoding the following peptide (a) or (b):
(a) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having JC virus particle-forming ability inhibitory activity; DNA consisting of the base sequence shown in SEQ ID NO: 1 or a complementary sequence thereof, and (5) a peptide that hybridizes with the DNA of claim 4 under stringent conditions and has JC virus particle-forming ability inhibitory activity The present invention relates to DNA encoding

The present invention also relates to (6) a peptide consisting of the amino acid sequence represented by SEQ ID NO: 4 and (7) an amino acid wherein one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4. A peptide comprising a sequence and having a particle-forming ability inhibitory activity of JC virus, and (8) a DNA encoding the following peptide (a) or (b):
(a) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4
(b) a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 and having a particle-forming ability inhibitory activity of JC virus, (9) A peptide comprising the nucleotide sequence shown in SEQ ID NO: 3 or a complementary sequence thereof, or (10) a peptide that hybridizes with the DNA according to claim 9 under stringent conditions and has JC virus particle formation inhibitory activity The present invention relates to DNA encoding

  Furthermore, the present invention provides (11) a fusion peptide in which the peptide according to claim 1 or 2 or 6 or 7 is bound to a marker protein and / or a peptide tag, or (12) claims 3 to 5 and 8 to 10. A JC virus particle formation inhibitor comprising the recombinant vector capable of expressing the DNA according to any one of 10 or (13) the peptide according to claim 1 or 2 or 6 or 7 as an active ingredient Or (14) a method for inhibiting JC virus particle formation, comprising administering the peptide according to claim 1 or 2 or 6 or 7 into a living body, or (15) claim 1 or 2 or 6 or A prophylactic / therapeutic agent for human progressive multifocal leukoencephalopathy characterized by comprising the peptide according to claim 7 as an active ingredient, or (16) the claim 1 or 2 or 6 or 7 A method for preventing or treating human progressive multifocal leukoencephalopathy characterized by administering a peptide into a living body, or (17) JC comprising the recombinant vector according to claim 12 as an active ingredient A virus particle formation inhibitor; (18) a method for inhibiting JC virus particle formation, comprising administering the recombinant vector according to claim 12 into a living body; and (19) a set according to claim 12. A prophylactic / therapeutic agent for human progressive multifocal leukoencephalopathy characterized by comprising a replacement vector as an active ingredient, and (20) a recombinant vector according to claim 12 administered in vivo The present invention relates to a method for preventing and treating human progressive multifocal leukoencephalopathy.

  According to the present invention, the formation of JCV particles can be inhibited, and as a result, the proliferation of the virus can be significantly suppressed, and a prophylactic / therapeutic agent for human progressive multifocal leukoencephalopathy becomes possible. That is, a therapeutic method that suppresses virus growth by inhibiting particle formation using a peptide-like compound having a JCV Agnoprotein partial sequence (viral particle formation inhibitory sequence) as an antiviral agent is replaced with a conventional therapeutic method. It can be provided as an effective treatment for PML. Since the virus particle formation inhibitory sequence of JCV has been clarified by the present invention, the most important partial structure for virus particle formation can be determined from the sequence. On the other hand, in the treatment method that blocks the mechanism involved in transcription and translation of the JCV gene, there are concerns about side effects on the host because many enzyme groups derived from the host are involved in the amplification of the JCV gene. However, in the treatment method according to the present invention, the mechanism of the virus itself called virus particle formation is blocked by a virus-derived compound, and more than 70% of humans are invisibly infected with JCV (ie, virus particles). From the viewpoint that the immune response to the formation-inhibiting sequence is considered to be small), there is an advantage that there are few side effects on the host cell. In addition, since DNA viruses have a lower frequency of mutation than RNA viruses such as HIV, it is considered that the influence of viral gene mutation is small. In particular, in the region encoding the JCV constituent protein, the degree of gene mutation is extremely low, and thus the possibility that a resistant virus appears is very low. Although it is necessary to efficiently introduce a compound having a virus particle formation inhibitory sequence into a disease site, most peptide-like compounds are water-soluble and can be easily administered to a living body. In addition, since peptide peptide synthesis methods have already been established, a large amount of peptide compounds can be synthesized in a short time.

  The peptide of the present invention includes (A) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4; (B) one or several amino acids deleted or substituted in the amino acid sequence shown in SEQ ID NO: 2 or 4 Alternatively, the peptide of the present invention is not particularly limited as long as it is a peptide consisting of an added amino acid sequence and having particle formation ability inhibitory activity of JC virus. The DNA of the present invention includes (A) the amino acid represented by SEQ ID NO: 2 or 4 DNA consisting of a nucleotide sequence encoding a peptide consisting of a sequence; (B) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and JC DNA comprising a nucleotide sequence encoding a peptide having virus particle-forming ability inhibitory activity; (C) shown in SEQ ID NO: 1 or 3 DNA consisting of a base sequence or a complementary sequence thereof; (D) hybridizing under stringent conditions with a DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3 or a complementary sequence thereof, and forming JC virus particles As long as it is DNA encoding a peptide having an activity inhibitory activity, it is not particularly limited, and “the peptide having JC virus particle formation inhibitory activity” herein refers to the evaluation of the ability to form VP1-derived virus-like particles in the Examples described later. In the above, a peptide in which the decrease in the amount of particle formation is statistically significant at P <0.05 as compared to the case in the absence of the peptide.

  The DNA consisting of the base sequence shown in SEQ ID NO: 1 includes the 31st to 90th base sequences of the DNA sequence encoding Agnoprotein shown in SEQ ID NO: 5, and the DNA consisting of the base sequence shown in SEQ ID NO: 3. As the amino acid sequence of Agnoprotein shown in SEQ ID NO: 6 as the peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 from the 142nd to 213th base sequence of the DNA sequence encoding Agnoprotein shown in SEQ ID NO: 5 Among them, the 11th to 30th amino acid sequences, and the peptide consisting of the amino acid sequence shown in SEQ ID NO: 4, include the 48th to 71st amino acid sequences in the amino acid sequence of Agnoprotein shown in SEQ ID NO: 6, respectively. Can do.

  The above “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3, further preferably 1 to 2. It means an amino acid sequence in which any number of amino acids are deleted, substituted or added. And a base encoding a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4, and having a particle forming ability inhibitory activity of JC virus As the DNA comprising the sequence, a DNA comprising a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 or 3 can be preferably exemplified. Exemplifying DNA consisting of a base sequence in which any number of bases of -20, preferably 1-15, more preferably 1-10, and even more preferably 1-5 are deleted, substituted or added. Can do.

  For example, DNA (mutant DNA) comprising a base sequence in which one or several bases are deleted, substituted or added can be obtained by any method known to those skilled in the art such as chemical synthesis, DNA engineering techniques, mutagenesis, etc. Can also be produced. Specifically, these DNAs can be obtained by using a method of bringing a DNA having the base sequence shown in SEQ ID NO: 1 or 3 into contact with a mutagen agent, a method of irradiating ultraviolet rays, a DNA engineering method, or the like. Mutant DNA can be obtained by introducing a mutation into. Site-directed mutagenesis, which is one of DNA engineering methods, is useful because it can introduce a specific mutation at a specific position. Molecular Cloning: A laboratory Mannual, 2nd Ed., Cold Spring Harbor Laboratory , Cold Spring Harbor, NY., 1989. (hereinafter abbreviated as "Molecular Cloning 2nd Edition"), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), etc. It can be carried out. By expressing this mutant DNA using an appropriate expression system, a peptide having an amino acid sequence in which one or several amino acids are deleted, substituted, or added can be obtained.

  The above-mentioned “base sequence that hybridizes under stringent conditions” means that a nucleic acid such as DNA or RNA is used as a probe, and a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like is used. Specifically, using a filter in which DNA derived from colonies or plaques or a fragment of the DNA is immobilized, the DNA is high at 65 ° C. in the presence of 0.7 to 1.0 M NaCl. After hybridization, the filter is washed under conditions of 65 ° C. using an SSC solution of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). The DNA which can be identified can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.

  For example, DNA that can hybridize under stringent conditions can include DNA having a certain degree of homology with the base sequence of the DNA used as a probe, for example, 60% or more, preferably 70%. Preferred examples thereof include DNA having homology of 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.

  The method for obtaining and preparing the DNA of the present invention is not particularly limited, and includes the nucleotide sequence information shown in SEQ ID NO: 1 or 3 disclosed in the present specification or the amino acid sequence information shown in SEQ ID NO: 2 or 4. Based on this, suitable probes and primers are prepared, and the target DNA is isolated by screening a cDNA library in which the DNA is expected to exist, or prepared by chemical synthesis according to a conventional method. can do.

  Specifically, by preparing a cDNA library from JCV derived from Agnoprotein according to a conventional method, and then selecting a desired clone from this library using an appropriate probe specific to the DNA of the present invention, The DNA of the present invention can be obtained, and the method for screening the DNA of the present invention from a cDNA library can include methods commonly used by those skilled in the art, such as the method described in Molecular Cloning 2nd edition. .

  The method for obtaining and preparing the peptide of the present invention is not particularly limited, and may be either a chemically synthesized peptide or a recombinant peptide produced by a DNA recombination technique. When preparing a peptide by chemical synthesis, for example, the peptide of the present invention can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). it can. Moreover, the peptide of this invention can also be synthesize | combined using various commercially available peptide synthesizers. When a peptide is prepared by a DNA recombination technique, the peptide of the present invention can be prepared by introducing DNA consisting of a base sequence encoding the peptide into a suitable expression system.

  For example, when preparing the peptides of the present invention by DNA recombination techniques, such peptides can be recovered from cell culture and purified by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography. , Known methods including hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, preferably high performance liquid chromatography. In particular, as a column used for affinity chromatography, for example, a column in which an antibody such as a monoclonal antibody against the peptide of the present invention is bound, or when a normal peptide tag is added to the peptide of the present invention, the peptide tag By using a column to which an affinity substance is bound, purified products of these peptides can be obtained.

  Furthermore, the peptide consisting of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 4 is an example of a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 Those skilled in the art can appropriately prepare or obtain the nucleotide sequence based on the information of the base sequence shown in SEQ ID NO: 1. For example, the DNA having the nucleotide sequence shown in SEQ ID NO: 1 or a part thereof can be used as a probe to isolate a DNA homolog by screening under appropriate conditions from an organism other than JCV. After cloning the full-length DNA of this homologous DNA, the DNA represented by SEQ ID NO: 1 or 3 is obtained by a conventional method, and this is incorporated into an expression vector and expressed in a suitable host, whereby the peptide encoded by the homologous DNA is obtained. Can be manufactured.

  The fusion peptide of the present invention in which the peptide of the present invention is bound to the marker protein and / or peptide tag is not particularly limited, and the marker protein is not particularly limited as long as it is a conventionally known marker protein. For example, enzymes such as alkaline phosphatase and HRP, Fc regions of antibodies, fluorescent substances such as GFP, etc. can be specifically mentioned. Examples of peptide tags in the present invention include HA, FLAG and Myc. Specific examples of known peptide tags such as epitope tags, affinity tags such as GST, maltose-binding peptide, biotinylated peptide, oligohistidine, and the like can be given. Such a fusion peptide can be prepared by a conventional method. Purification of the peptide of the present invention utilizing the affinity between Ni-NTA and His tag, detection of the peptide of the present invention, and quantification of an antibody against the peptide of the present invention It is also useful as a research reagent in this field.

  The recombinant vector of the present invention is not particularly limited as long as it is capable of expressing the peptide of the present invention by appropriately integrating the DNA of the present invention into an expression vector for animal cells. Such an expression vector is preferably one that can replicate autonomously in a host cell, or one that can be integrated into the chromosome of the host cell, and a promoter, enhancer, or terminator at a position where the DNA of the present invention can be expressed. Those containing a control sequence such as can be suitably used.

  As expression vectors for animal cells, for example, EGFP-C1 (Clontech), pGBT-9 (Clontech), pcDNAI (Funakoshi), pcDM8 (Funakoshi), pAGE107 (Cytotechnology, 3, 133, 1990), pCDM8 (Nature, 329, 840, 1987), pcDNAI / AmP (Invitrogen), pREP4 (Invitrogen), pAGE103 (J. Blochem., 101, 1307, 1987), pAGE210, etc. Can do. Examples of promoters for animal cells include cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter and the like. Can do.

  The particle formation inhibitor of the JC virus of the present invention is not particularly limited as long as it is a composition comprising the peptide of the present invention or the recombinant vector of the present invention as an active ingredient, and the human progressive multifocality of the present invention. The prophylactic / therapeutic agent for leukoencephalopathy (PML) is not particularly limited as long as it is a composition comprising the peptide of the present invention or the recombinant vector of the present invention as an active ingredient. , Pharmaceutically acceptable conventional carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegrating agents, solubilizers, solubilizers, isotonic agents, and various other formulation ingredients Can be added. These prophylactic / therapeutic agents for PML can be administered orally or parenterally, and can be administered orally, for example, in the form of powder, granules, tablets, capsules, syrups, suspensions, etc. Alternatively, for example, in the form of a solution, emulsion, suspension or the like, it can be administered parenterally in the form of injection, or it can be administered intranasally in the form of spray. Moreover, the dosage of the prophylactic / therapeutic agent for PML of the present invention can be appropriately selected according to the weight and age of the patient, the dosage form, symptoms and the like.

  In the case of preparations for oral administration, various conventional organic or inorganic carrier substances are used as pharmacologically acceptable carriers. For example, tablets include excipients such as lactose and starch, talc, and magnesium stearate. Lubricants such as hydroxypropylcellulose and polyvinylpyrrolidone, disintegrants such as carboxymethylcellulose, etc. can be blended. Solvents such as physiological saline alcohol, polyethylene glycol, propylene Solubilizing agents such as glycol, suspending agents such as stearyltriethanolamine, sodium lauryl sulfate, lecithin, isotonic agents such as glycerin and D-mannitol, buffering agents such as phosphate, acetate and citrate Etc. can be blended. If necessary, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be blended. For preparations for parenteral administration, water-soluble solvents such as distilled water and physiological saline, solubilizing agents such as sodium salicylate, isotonic agents such as sodium chloride, glycerin and D-mannitol, human serum albumin, etc. Stabilizers, preservatives such as methylparaben, and narcotics such as benzyl alcohol can be blended.

  The method for inhibiting particle formation of the JC virus of the present invention is not particularly limited as long as it is a method for in vivo administration of the peptide of the present invention or the recombinant vector of the present invention, and the human progressive multifocal of the present invention. The method for preventing and treating leukoencephalopathy (PML) is not particularly limited as long as it is a method of administering the peptide of the present invention or the recombinant vector of the present invention into a living body. As a method for administering the recombinant vector of the present invention into a living body and delivering it into a cell, a method for introducing the above-described recombinant vector of the present invention into a cell, or a method for introducing the above-mentioned peptide of the present invention into a cell. There is no particular limitation as long as the recombinant vector is introduced into the cell, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transbex (Transvection), microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection, etc., and the peptide of the present invention Chariot (Active Motif) can form macromolecules such as proteins by changing the structure of macromolecules such as proteins and delivering macromolecules such as proteins into cells. And a non-cytotoxic reagent such as a liposome-like cationic lipid that forms a complex non-covalently with a peptide and is taken into the cell.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Confirmation of binding of outer shell protein VP1 and Agnoprotein-derived peptide by Overlay method]
JCV is a double-stranded circular DNA virus of 5,130 bp, and the genomic DNA of the virus is contained (packaged) in the outer shell proteins VP1, VP2, and VP3. On the other hand, Agnoprotein consists of 71 amino acids, and the inventors have shown that it binds to VP1, but it has not been clarified which region of 71 amino acids is necessary for binding. Therefore, the inventors synthesized eight types of peptide fragments consisting of about 20 to 30 residues derived from Agnoprotein, and analyzed whether or not this and VP1 bind to each other using the Overlay method. Specifically, after 10 μg of the peptide is spotted on a nitrocellulose membrane and air-dried, the peptide on the membrane, the VP1 N-terminal half or C-terminal half region, and a glutathione-S-transferase (GST) fusion protein Reacted. The VP1 binding region was determined by detecting the bound GST fusion proteins (referred to as GST-VP1 (N) and GST-VP1 (C), respectively) with an anti-VP1 antibody.
[Determination of binding region of VP1 and Agnoprotein]
Eight kinds of synthetic peptide fragments used in the experiment are shown in FIG. Of these peptide fragments, it was shown that the 11th to 38th region peptides of Agnoprotein bind to both the N- and C-terminal regions of VP1 (FIG. 2). Moreover, it was shown that the 21st to 38th peptides bind to the N-terminal region of VP1, and the 11th to 30th and 41st to 71st peptides bind to the C-terminal region of VP1 (FIG. 2). None of the peptides bound to GST alone.

[Evaluation of VP1-derived virus-like particle-forming ability in the presence of Agnoprotein-derived peptide]
(1) Gel filtration method and (2) 5% to 35% sucrose density gradient centrifugation were used to evaluate the ability to form VP1-derived virus-like particles. Each 2 μM VP1 and an Agnoprotein-derived synthetic peptide were mixed and subjected to particle formation reaction on ice for 1 hour, and then the amount of particle formation was evaluated by gel filtration and sucrose density gradient centrifugation. The gel filtration method can separate a VP1-derived virus-like particle, a VP1 monomer, and a VP1 pentamer as an intermediate thereof. Using this fact, the peak intensity derived from the VP1-derived virus-like particles was measured, and the amount of virus-like particles formed in the presence of each peptide was measured. In the sucrose density gradient centrifugation, virus-like particles and VP1 monomer can be separated by the difference in specific gravity, and virus-like particles are precipitated in the fraction with the highest specific gravity. The solution after centrifugation was fractionated by 1 ml, VP1 in the fraction was detected with an anti-VP1 antibody, and the influence of each peptide on the ability to form virus-like particles was evaluated.
[Effect of Agnoprotein-derived peptide on virus-like particle formation]
FIG. 3 shows the results of the gel filtration method in which VP1 was synthesized using E. coli and the amount of virus-like particles having the same form as virus and the same cell invasion ability was examined. Assuming that the amount of particle formation in the absence of peptide is 1, the amount of particle formation is reduced to about half in the presence of the 11th to 30th peptides. On the other hand, it increased about 2-fold in the presence of the 41st to 71st peptides. These decreases and increases were statistically significant at P <0.05 (* mark). In addition, in the presence of the 11th to 38th, 21th to 38th, and 48th to 71st peptides, not only the peak of the particles, but also the peak of the VP1 pentamer or monomer was observed. The amount of virus-like particle formation could not be measured by this method (N / A in the figure). Then, the result of having evaluated the influence which each peptide has on the particle formation amount by sucrose density gradient centrifugation is shown in FIG. In the absence of peptide (Control in the figure), VP1 was widely detected from a fraction with a low specific gravity (Top in the figure) to a heavy fraction (Bottom in the figure). In particular, VP1 detected in the heaviest fraction was considered to be particles. In this fraction, a signal of VP1 was detected even in the presence of six peptides from 1 to 20, 11 to 38, 21 to 38, 31 to 60, 41 to 71, and 41 to 60. However, no signal indicating particle formation was detected in the presence of the 21st to 38th and 48th to 71st peptides. From this, it was considered that the 21st to 38th and 48th to 71st peptides were particle formation inhibiting sequences.

It is a figure which shows 8 types of synthetic peptide fragments derived from Agnoprotein used for the Example. It is shown that the peptides in the 11th to 38th regions of Agnoprotein shown in FIG. 1 bind to both the N- and C-terminal regions of VP1, and that the 21st to 38th peptides bind to the N-terminal region of VP1. It is a figure which shows that two types of peptides, 11th to 30th and 41st to 71st, bind to the C-terminal region of VP1. It is a figure which shows the result of having investigated the virus-like particle formation amount of VP1 synthesize | combined using colon_bacillus | E._coli by the gel filtration method. It is a figure which shows the result of having evaluated the influence which the peptide of this invention has on particle formation amount by the sucrose density gradient centrifugation method.

Claims (20)

A peptide consisting of the amino acid sequence shown in SEQ ID NO: 2. A peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having JC virus particle-forming ability inhibitory activity. DNA encoding the following peptide (a) or (b).
(a) a peptide comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having an activity of inhibiting particle formation ability of JC virus DNA consisting of the base sequence shown in SEQ ID NO: 1 or its complementary sequence. A DNA that hybridizes with the DNA of claim 4 under stringent conditions and encodes a peptide having JC virus particle-forming ability inhibitory activity. A peptide consisting of the amino acid sequence shown in SEQ ID NO: 4. A peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4, and having an activity of inhibiting particle formation ability of JC virus. DNA encoding the following peptide (a) or (b).
(a) a peptide consisting of the amino acid sequence shown in SEQ ID NO: 4
(b) a peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4 and having an activity of inhibiting particle formation ability of JC virus DNA consisting of the base sequence shown in SEQ ID NO: 3 or its complementary sequence. A DNA that hybridizes with the DNA of claim 9 under stringent conditions and encodes a peptide having JC virus particle-forming ability inhibitory activity. A fusion peptide in which the peptide according to claim 1 or 2 or 6 or 7 is bound to a marker protein and / or a peptide tag. A recombinant vector capable of expressing the DNA according to any one of claims 3 to 5 and 8 to 10. A JC virus particle formation inhibitor comprising the peptide according to claim 1 or 2 or 6 or 7 as an active ingredient. A method for inhibiting JC virus particle formation, comprising administering the peptide according to claim 1 or 2 or 6 or 7 into a living body. A prophylactic / therapeutic agent for human progressive multifocal leukoencephalopathy, comprising the peptide according to claim 1 or 2 or 6 or 7 as an active ingredient. A method for preventing / treating human progressive multifocal leukoencephalopathy, comprising administering the peptide according to claim 1 or 2 or 6 or 7 into a living body. 13. A particle formation inhibitor of JC virus comprising the recombinant vector according to claim 12 as an active ingredient. A method for inhibiting particle formation of JC virus, which comprises administering the recombinant vector according to claim 12 into a living body. A prophylactic / therapeutic agent for human progressive multifocal leukoencephalopathy comprising the recombinant vector according to claim 12 as an active ingredient. A method for preventing / treating human progressive multifocal leukoencephalopathy, comprising administering the recombinant vector according to claim 12 into a living body.

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