JP2008247864A - Mosaic serine protease, msp and its use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To promote a safer and more effective antiviral agent, and basic and applied research on highly pathogenic avian influenza, HPAI, and to establish a method for diagnosing abnormal production of a peptide hormone related to MSP under a situation that no vaccine nor drug having specific effectiveness against HPAI are practically used and an existing drug causes a serious side effect. <P>SOLUTION: MSPL, MSPS and TMPRSS13 inhibitors belonging to mosaic serine protease, MSP; a screening method for the above inhibitor using the degree of decomposition or cleavage of a substrate caused by these enzymes as an index; an antivirus agent and a therapeutic agent for peptide hormone disorder having the inhibitor as an active ingredient; an activation agent for enhancing infection and propagation of an HPAI virus and enabling its mass production; a therapeutic agent for peptide hormone disorder having the above enzymes as an active ingredient; an agent for diagnosing a hormone disorder using the amount of genetic expression of the above enzymes as an index; and the like. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a mosaic serine protease and its use. More specifically, an antiviral agent using the protease substrate specificity, inhibitor spectrum, gene expression, and the like as an index and a screening method thereof, particularly an antiviral agent for the treatment or prevention of severe pathogenic avian (bird) influenza. Virus agents and screening methods thereof, influenza virus infection or proliferation activators, virus particles produced by using the activators and their constituents, hormone production abnormal diagnostic agents and therapeutic agents, etc. is there.

<Explanation of abbreviations>
MSP: Mosaic serine protease; MSPL: MSP long chain (mosaic serine protease, long); MSPL: MSP short chain (mosaic serine protease, short); TMPRSS13: transmembrane serine protease 13b HA: hemagglutinin; HPAI: highly pathogenic avian influenza; RT-PCR: reverse-transcription polymerase reaction.
<Description of sequence number>
Each of the following sequences is the latest revised version as of March 30, 2007, at the time of filing this patent, of the sequence described in Non-Patent Document 1 described later.
SEQ ID NO: 1 shows the base sequence of the full-length codon region of MSPL gene cDNA.
SEQ ID NO: 2 shows the full-length amino acid sequence of MSPL.
SEQ ID NO: 3 shows the base sequence of the full-length codon region of MSPS gene cDNA.
SEQ ID NO: 4 shows the full length amino acid sequence of MSPS.
SEQ ID NO: 5 shows the base sequence of the full-length codon region of TMPRSS13 gene cDNA.
SEQ ID NO: 6 shows the full-length amino acid sequence of TMPRSS13.

The first inventors cloned two new MSP genes from a cDNA library derived from human lung, determined the base sequence of each of these genes, decoded the amino acid sequence encoded by them, The peptide chain was analyzed for structure, etc. and named MSPL (GenBank Accession: AB048796) and MSPS (Accession: AB048797), respectively, and reported in 2001 (Non-patent Document 1). Thereafter, TMPRSS13 (Accession: NM_001077263) was discovered from the search for homology with these enzymes. However, reports and public documents that clarify the functions of these enzymes are still unknown.
Regarding the enzyme MSP and its gene, and their use, the following techniques are known: The first inventors use “MSP and its gene” (Patent Document 1) and “MSP activity inhibition of the invention as an index. Screening method for anti-influenza drugs "(Patent Document 2), regulator of MSP production in the body (Patent Document 3), metabolic activator using MSP and its gene (Patent Document 4), diagnostic agent for various diseases involving MSP And therapeutic agents (Patent Documents 5 and 6).
Vaccines and anti-influenza drugs have been put into practical use for the prevention and treatment of influenza. The following drugs are clinically used as anti-influenza drugs: amantadine hydrochloride (trade name: symmetril, orally administered, acts on M2 protein to inhibit viral shelling, and is effective against influenza A, but M2 Not effective for type B without side effects, adverse effects on central nervous system such as insomnia, loss of appetite, nausea, etc., zanamivir (trade name Relenza, oral inhalation administration, infection of influenza virus from cell to cell by neuraminidase inhibition・ Effective for both A and B types to prevent transmission, side effects rarely occur anaphylaxis-like symptoms, urticaria, asthma attack, etc., and oseltamivir phosphate (trade name Tamiflu, oral administration, action is neuraminidase Same as zanamivir, effective for both A and B types, side effects include abdominal pain, diarrhea, nausea, and psychiatric nerve Jo, anaphylactic-like symptoms, acute renal failure, etc.).
In addition, there are already a wide variety of reports, knowledge, techniques, etc. concerning the cleavage of influenza virus nucleosylkabside by protease, analysis of the site, cleavage inhibitor (protease inhibitor), correlation between such cleavage and infection, etc. Known to. For example, cleavage by furin (endoprosthesis) (Non-patent document 2), cleavage of HIV by serine protease and separation of its fragment peptide (non-patent document 3), necessity of miniplasmin in influenza virus infection (non-patent document 4) ), Protease is essential for the entry of influenza virus into cells (Non-patent Document 5), and so on.
In addition, in this invention, the technique described in these literature is used as a conventional method in description of the experiment example and Example which are mentioned later.

JP2000-253887 JP 2001-69980 A WO 01/96538 WO 03/093432 WO 2005/040401 US Publication No. US2006 / 0292155 (US application of Patent Document 5) Biochemica et Biophysica Acta, 1518, 204-209, 2001. EMBO Journal, 11 (7), 2407-2414, 1992 European Journal of Biochemistry, 237, 64-74, 1996. European Journal of Biochemistry, 268, 2847-2855, 2001. Current Pharmaceutical Design, 13, 403-412, 2007.

Although the original host of HPAI virus is a bird and it is difficult to infect humans, neither a vaccine nor a drug specifically effective against highly pathogenic avian (avian) influenza has been put into practical use. In addition, current anti-influenza drugs, although rare, cause serious side effects. Therefore, development of safer and more effective drugs, further progress in virology and vaccinology related to HPAI and applied research, etc. It is the current situation that is eagerly expected. In addition, establishment of a method for diagnosing abnormal hormone production involving MSP is also an important issue.

As described above, since the first inventors reported the discovery of two enzymes, MSPL and MSPS, in 2001 (Non-Patent Document 1), they discovered TMPRSS13 as a homologous enzyme. As a result of repeated research on the function, the surprising function that these enzymes cleave or cleave the highly pathogenic avian (avian) influenza, that is, the HA peptide chain of the HPAI virus very specifically and efficiently. discovered. The present invention has been conceived as a result of such discovery, and has been completed as a result of further ingenuity, trial and error, and diligence.

According to the present invention, the following (1) to (9) are provided as means for solving the above-described problems:
(1) An inhibitor for at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13.
(2) A screening method for an enzyme inhibitor, characterized by using, as an index, the degree of degradation or cleavage of a substrate by at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13.
(3) An enzyme inhibitor obtained by the above screening method.
(4) An antiviral agent comprising the above-mentioned (1) or (3) inhibitor or enzyme inhibitor as an active ingredient in an effective amount.
(5) A virus infection and / or proliferation activator containing an amount having medicinal effects, comprising at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13 as an active ingredient.
(6) Virus particles produced by using the activator of (5) above and / or components thereof.
(7) A therapeutic drug for a hormonal disease containing an effective amount of at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13 as an active ingredient.
(8) A therapeutic drug for a hormonal disease containing an effective amount of an inhibitor for at least one enzyme selected from the enzyme group consisting of MSPL, MSPS and TMPRSS13 as an active ingredient.
(9) A diagnostic agent for hormonal diseases using the gene expression level of at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13 as an index.

(1) A safe and effective antiviral agent that functions extremely specifically in the treatment and prevention of serious infections of virus infections such as new influenza and AIDS, which are derived from HPAI and have a death rate due to infection exceeding 30%, and a screening method therefor Is provided.
(2) A therapeutic and diagnostic agent for a disease caused by abnormal production of peptide hormones is provided.
(3) The virus infection and / or proliferation activator provided by the present invention is a mass production of viruses that are difficult to culture or mass-produce, such as virus particles of avian influenza and its components, virus antigens, virus genes, antibodies, etc. It will be useful for the further development of basic research and applied research in the fields of virology and vaccinology.
(4) The above (1) to (3) greatly contribute to the progress of human health and civilization, and immeasurable mental effects on various activities such as private life, social life, business activities, academic activities, etc. And bring about economic effects.

The embodiment of the present invention will be described in detail as follows:
(1) Target diseases of antiviral agents Viral diseases targeted by the antiviral agents according to the present invention are basics such as KR, RR, RKR, RRRR, two or more R (arginine), K (lysine), etc. It is an infectious disease caused by a series of viruses having a nucleocapsid in which a primary structure in which amino acids are continuously peptide-bonded is present, and the cleavage or cleavage of the nucleocapsid is involved in pathogenic expression.
For example, a novel influenza virus originated from HPAI, mumps virus, canine distemper virus, RS virus (respiratory synchronous virus), yellow fever virus, HIV (human immunodefectious virus), human cytomegal virus, varicella virus, toxic NDV Infectious diseases such as Newcastle disease virus) can be raised.

(2) Hormonal disease The hormonal disease according to the present invention is a primary structure in which two or more basic amino acids such as R (arginine) and K (lysine), such as KR, RR, RKR, RRRR, are continuously peptide-bonded. Means a disease caused by abnormal production of peptide hormones that are activated by cleaving or cleaving the peptide hormone precursor present in the presence of at least one enzyme selected from the enzyme group consisting of MSPL, MSPS and TMPRSS13 To do.
For example, diseases caused by abnormal production of peptide hormones such as renin, von Willebrand factor, b-nerve growth factor can be raised.

(3) Inhibitor or Enzyme Inhibitor The inhibitor or enzyme inhibitor according to the invention is a polyclonal antibody against MSPL, MSPS and TMPRSS13, epitopes of these enzymes, ie, SEQ ID NOs: 2, 4 and 6 Monoclonal antibodies against epitopes selected from amino acid sequences, genes encoding these enzymes, that is, siRNAs designed from the cDNA base sequences set forth in SEQ ID NOs: 1, 3, and 5, compounds extracted and purified from natural products, It means a synthetic compound. Inhibitors used as therapeutic agents for viral infections and hormonal diseases are preferably low molecular weight compounds having no immunogenicity or antigenicity in consideration of repeated use and parenteral administration. Specific examples of inhibitors at the experimental stage are listed in Table 2.

(4) Enzyme In the present invention, MSPL, MSPS and TMPRSS13 are used as enzymes, respectively. These enzymes can be mass-produced by conventional methods of gene recombination. For example, after constructing an expression vector for each enzyme gene, the vector can be mass-produced by culturing transformants obtained by transferring the vectors to hosts such as human cells, yeast, E. coli, etc. Specific examples thereof are described in Experimental Example 3.
The structures of these enzyme genes are shown in FIGS. 1 to 3, the amino acid sequence comparison is shown in FIG. 4, and the structure of MSPL is shown in FIG.
The expression levels of the enzyme in various human tissues are shown in FIG. 6 as the results of Experimental Example 2, respectively.

(5) Substrate In consideration of the substrate specificity of the enzyme of the above (4) according to the present invention, two or more basic amino acids such as R (arginine) and K (lysine) such as KR, RR, RKR, RRRR are present. It is desirable to use peptides in which there is a continuous primary structure with peptide bonds. Specific examples thereof are listed in Tables 1 and 3. In addition, the substrate used for screening for inhibitors as therapeutic drug candidates can be appropriately selected according to the purpose. For example, for screening antiviral agents against HPAI virus, the virus's own HA is used as a substrate. For the screening of therapeutic agents for peptide hormone diseases, the precursor of the hormone is used as a substrate.

(6) Measurement of enzyme activity The measurement is performed by an enzyme reaction method and a conventional method for the measurement. In other words, after mixing an enzyme and a substrate at a constant concentration, the enzyme composition is subjected to an enzyme reaction for a certain period of time under the conditions of the solvent composition, its pH, and temperature, and then measured by quantifying the enzyme degradation products. To do. A specific example is described in Experimental Example 4.

(7) Substrate specificity of enzyme Substrate specificity can be determined by measuring the degradation activity of various substrates by an enzyme in the same manner as the measurement of enzyme activity described above. Specific examples thereof are described in Example 1.

(8) Specificity of enzymes for various inhibitors (inhibitor spectrum)
The inhibitor spectrum is assayed by performing an enzyme reaction in the presence of an inhibitor. After mixing the enzyme and the inhibitor, the mixture can be assayed by conducting an enzyme reaction in the same manner as the measurement of the enzyme activity described above and quantifying the degree of degradation of the substrate or the remaining degree of the undegraded substrate. it can. A specific example is described in Example 2.

(9) Screening method for antiviral agents Antiviral agents are screened based on the above inhibitor spectrum. In this case, the substrate is a virus-constituting protein prepared from the target pathogen virus itself, and a substance having a high substrate specificity of the enzyme according to the present invention, such as nucleocapsid, capsomere, HA, etc. in the case of HPAI virus. Is desirable. As described above, as a screening inhibitor candidate substance, it is desirable to use a low molecular weight compound having no immunogenicity or antigenicity. A specific example is described in Example 3.

(10) Activator of virus infection and / or growth Regarding the activator according to the present invention, the aforementioned enzymes MSPL, MSPS and TMPRSS13 are used as active ingredients of the activator. This activator is capable of mass production of all components of viruses such as HPAI virus and HIV, which are difficult to culture and mass-produce, and viral genes, antigens, viral enzymes, structural proteins, nucleocapsids, capsomeres, etc. Enables and facilitates the production of antibodies against antigens. The activator can be used, for example, in the form of addition and mixing to a virus culture medium or addition and mixing of a seed virus to a pretreatment solution.

(11) Therapeutic Agent for Peptide Hormone Disease When the disease is caused by excessive production of peptide hormone, an inhibitor screened and provided by the present invention can be used as the therapeutic agent. Conversely, when the production of the hormone is insufficient, the enzyme according to the present invention can be used as a replacement therapy agent.
Hereinafter, the configuration and effects of the present invention will be specifically described with reference to experimental examples and examples. However, the present invention is not limited only to these experimental examples and examples.

[Experimental Example 1]
Cloning, expression and analysis of each gene of MSPL, MDPS and TMPRSS13 Each of the above genes was cloned by PCR from a human lung cDNA library [trade name Marathon-Ready-cDNA manufactured by Clontech (USA)] and then expressed respectively. It was. In addition, the base sequences of these gene cDNAs were determined, and the amino acid sequences encoded by them were decoded. The details of the materials and methods and the results are described in the previous report (Non-Patent Document 1) by the first inventors.
(1) Furthermore, as a result of reconfirming the description in the previous report, an error was found in the base sequence and amino acid sequence. Correction data based on the result is shown in FIGS. 1, 2, and 3, respectively.
FIG. 1 shows the base sequence of the full-length codon region of MSPL gene cDNA and the full-length amino acid sequence encoded by it. The amino acid surrounded by □ is the active center, ↑ is the cleavage site for becoming an active form, the underlined bold line is the putative transmembrane region, and * is the putative N-type glycosylation site.
FIG. 2 shows the base sequence of the full-length codon region of MSPS gene cDNA and the full-length amino acid sequence encoded by it. Amino acids surrounded by □ are active centers, ↑ is a cleavage site for becoming an active form, and * is a putative N-type glycosylation site.
FIG. 3 shows the base sequence of the full-length codon region of TMPRSS13 gene cDNA and the full-length amino acid sequence encoded by it. The amino acid surrounded by □ is the active center, ↑ is the cleavage site for becoming an active form, the underlined bold line is the putative transmembrane region, and * is the putative N-type glycosylation site.
(2) In addition, these three amino acid sequences were compared with each other. The result is shown in FIG. In this figure, the translation start site Met is the first amino acid, and the homologous site is surrounded by a square.
(3) Moreover, the structural analysis of MSPL was conducted. The result is shown in FIG. In this figure, the domain structure of MSPL is schematically shown.
Incidentally, TM is t rans m embrane domain (a transmembrane domain), SRCR is s cavenger r eceptor c istein- r ich domain ( scavenger receptor cysteine-rich domains), and the SPD s erine p rotease d omain (the serine protease domain) Each means.
Tandem means an amino acid sequence of tandem repeat sequence found in the N-terminal side intracellular region, and further, phosphorylation site recognition sequences by various kinases, that is, recognition sequences by Cdc2 kinase S / TPPX-K Recognized sequences of / R, R—X—X—S / T by CaM kinase II, and R—X—X—S—X—R by protein kinase C are shown in bold. Furthermore, the repeatedly recognized amino acid sequence ASPA is underlined.

[Experiment 2]
Analysis of MSPL and TMPRSS13 gene expression in various human tissues mRNA expression in human tissues of MSPL and TMPRSS13 was performed using RT-PCR (reverse transcription-) using Human Multiple Tissue cDNA panels [purchased from BD Biosciences (USA)]. (polymerase chain reaction). Gene-specific primers for PCR include 5′-GACCCTGTCCCGCTCACATCCACCCT-3 ′ for the sense strand common to MSPL and TMPRSS13, 5′-CCTCCTGCCCTACACCCTGGGTGCTCCT-3 ′ for MSPL and 5 for the antisense strand of TMPRSS13. '-CTGGTTAGGATTTTCTGAATCGCAC-3' was used, respectively. PCR was performed under the following conditions: MSPL denaturation at 94 ° C. for 30 seconds, annealing at 66 ° C. for 30 seconds, extension at 72 ° C. for 30 seconds for 35 cycles, and TMPRSS13 for denaturation at 94 ° C. for 30 cycles. Second, 35 cycles of annealing at 62 ° C for 30 seconds and extension at 72 ° C for 30 seconds. The internal standard includes human GAPDH (glyceraldehydes-3-phosphate dehydrogenase) specific primer, GAPDH sense chain 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3 ', GAPDH antisense chain 5'-CATGGTG-3' used. PCR was performed under the following conditions: 25 cycles of denaturation at 94 ° C for 30 seconds, annealing and extension at 68 ° C for 2 minutes. PCR products were electrophoresed using 2% agarose / TAE (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA) gels and detected by ethidium bromide staining. It was confirmed by sequencing that the PCR product was the target gene. The result is shown in FIG.
In FIG. 6, lane 1 is a control (no template), lane 2 is brain, lane 3 is heart, lane 4 is lung, lane 5 is thymus, lane 6 is liver, lane 7 is spleen, lane 8 is pancreas, lane 9 is kidney, lane 10 is small intestine, lane 11 is large intestine, lane 12 is prostate, lane 13 is testis, lane 14 is ovary, lane 15 is skeletal muscle, lane 16 is placenta, lane 17 is leukocyte or peripheral blood lymphocyte, Lane 18 is mononuclear cell, lane 19 is CD14 ⁺ cell, lane 20 is CD4 ⁺ cell, lane 21 is CD8 ⁺ cell, and lane 22 is CD19 ⁺ cell.
MSPL showed high gene expression in lung, pancreas, prostate, and placenta. TMPRSS13 showed high gene expression not only in lung, pancreas, prostate and placenta, but also in thymus, spleen and peripheral blood lymphocytes, as well as in brain and large intestine.
As for MSPS, RT-PCR using specific primers was performed as described above. As a result, high gene expression was observed not only in the lung, pancreas, prostate and placenta, but also in ovary and peripheral blood lymphocytes.

[Experiment 3]
Preparation of MSPL and TMPRSS13 (1) Construction of Recombinant MSPL and TMPRSS13 Expression Vectors After amplification by PCR using the full-length cDNAs of MSPL and TMPRSS13 as templates, the product was obtained from the expression vector p3XFLAG-CMV14 [SIGMA (USA) It was incorporated into restriction enzyme EcoRI / XbaI sites of [purchase] to construct an expression vector. At the same time, only the extracellular regions of MSPL and TMPRSS13 (from SRCR domain to serine protease domain) are incorporated directly under the enterokinase recognition sequence (DDDDDK) of the expression vector p3XFLAG-CMV9 [purchased from SIGMA (USA)]. An expression vector was constructed. Next, the sequences of all these expression vectors were confirmed by sequence analysis.
(2) Production and purification of recombinant MSPL and TMPRSS13 After each of the above expression vectors was transferred to HEK-293T cells (human embryonic kidney-derived cells) using Hily Max (purchased from Dojin Chemical Laboratory (Japan)). , it was cultured in serum-free medium, the resulting culture supernatant (SFCM; S erum f ree c ulture m edia) were collected. The supernatant was then concentrated using an ultrafiltration Biomax-10 membrane (purchased from Millipore (USA)), and then equilibrated with 50 mM Tris-HCl (pH 7.4) and 150 mM NaCl. Thereafter, it was applied to an anti-FLAG M2-agarose affinity gel column [purchased from SIGMA (USA)] and eluted with 0.1 M Glycine-HCl (pH 3.5) for purification. The obtained purified protein was dialyzed with Phosphate-buffered saline (PBS) and stored at −30 ° C. until use.
(3) Activation of MSPL and TMPRSS13 by trypsin 2 μg of recombinant MSPL was obtained from 10 μl of trypsin beads [immobilized Tos-Phe-CH ₂ Cl (tosylphenylally chloromethane) -trypsin] (Pirce, USA) The reaction was carried out at 37 ° C. for 2 hours in (pH 8.0). After completion of the reaction, trypsin beads were removed by centrifugation at 20,400 × g for 2 minutes, and then the supernatant was collected and its MSPL enzyme activity was confirmed by measuring by the method described in Experimental Example 4.

[Experimental Example 4]
Measurement of enzyme activity The enzyme activity was measured by reacting activated recombinant MSPL with a fluorescent-labeled artificial peptide substrate in Tris buffer (0.1 M Tris-HCl, pH 8.5), and the reaction product (AMC; 7- a mino-4 m ethyl c oumarin) fluorescence spectrophotometer production of using [HITACHI Ltd. (Japan) made Model 650-10MS], was measured at Excitation wavelength 370 nm, and Emission wavelength 460 nm. Enzyme activity was defined as 1 unit (Unit), which is the amount of enzyme that produces 1 μmol of AMC per minute.

Enzyme Substrate Specificity In the same manner as described in Experimental Example 4, the degradation activity of various substrates by the enzyme was measured. The obtained measured value was expressed as the degradation activity ratio (%: relative activity) of each substrate, with the degradation activity of the artificial substrate Boc-Gln-Ala-Arg-MCA being 100. The results are shown in Table 1.
In Table 1, Boc means t-butycarbonyl, MCA means 4-methylcoumeric-7-amide, Bz means benzoyl, and OBzl means benzyloxy.
MSPL is very specific for Boc-Leu-Arg-Arg-MCA, Boc-Glu-Arg-Arg-MCA, Boc-Leu-Lys-Arg-MCA, and Boc-Arg-Val-Arg-Arg-MCA Disassembled.

Specificity of enzymes for various inhibitors (inhibitor spectrum)
The influence of various inhibitors was determined by reacting activated recombinant MSPL and inhibitor in advance in a buffer solution at 37 ° C. for 5 minutes, and then measuring the remaining MSPL enzyme activity as described in Experimental Example 4. The obtained measured value is the ratio of the residual activity in the presence of each inhibitor (%: relative residual activity) where the degradation activity of the artificial substrate Boc-Gln-Ala-Arg-MCA in the absence of the inhibitor is taken as 100. The notation. The results are shown in Table 2.
In Table 2, E-64c is synthetic E-64 [trans-epoxysuccinyl-L-leucylamide- (4-guanidino) butane] analogue, UTI is urinary trypsin inhibitor, and SLPI is secure.
MSPL activity was specifically inhibited by Aprotinin, Benzamidine, Bowman-Birk trypsin inhibitor, and dec-RVKR-cmk.

Cleavage efficiency of hemagglutinin-restricted degradation site peptide of influenza virus by MSPL Following the cleavage efficiency of hemagglutinin-restricted degradation site peptide related to the expression of membrane fusion activity and infectivity of highly pathogenic avian influenza virus and human pathogenic influenza virus by MSPL Measured as follows.
After synthesizing peptides containing limited pathogenic sites for hemagglutinin of highly pathogenic avian influenza H5N1 and H7N7 and low pathogenic human influenza H3N2, 0.1 μg of MSPL was added to 10 μg of these peptides and mixed. And the peptide cleavage activity by MSPL was assayed. The reaction was carried out at 37 ° C. for 3 hours in 50 μl of 100 mM Tris buffer (Tris-HCl, PH 7.0). After completion of the reaction, trichloroacetic acid was added and mixed so as to be 1%, and then the reaction product was separated by reverse phase chromatography using ODS-120T by a conventional method (Non-patent Document 3). The amino acid sequence of the separated peptide was determined using an applied biosystems model 492 protein sequencer [Applied
Biosystems (USA)] was used to determine the cleavage site and the amount of the cleaved peptide. The cleavage rate (%) was expressed as the ratio of degradation products when the added peptide amount (mol) was 100. The results are shown in Table 3.
MSPL hardly cleaves the peptide structure of the low pathogenic human influenza H3N2 hemagglutinin, and it was confirmed that both the peptide structures of both the highly pathogenic avian influenza H5N1 and H7N7 maglutinins were cleaved very specifically.

  The present invention relates to antiviral agents and screening thereof, viral infection and / or proliferation activators, therapeutic agents for peptide hormone diseases and screening thereof, diagnostic agents, etc. It can be used in fields such as diagnostic agents and veterinary medicine.

FIG. 1 shows the nucleotide sequence and amino acid sequence of MSPL. FIG. 2 shows the nucleotide sequence and amino acid sequence of MSPS. FIG. 3 shows the nucleotide sequence and amino acid sequence of TMPRSS13. FIG. 4 shows an amino acid sequence comparison of MSPL, MSPS and TMPRSS13. FIG. 5 shows the structure of MSPL. FIG. 6 shows a comparison of gene expression of MSPL and TMPRSS13 in human tissues.

Claims (9)

An inhibitor for at least one enzyme selected from the group consisting of MSPL, MSPS, and TMPRSS13. A screening method for an enzyme inhibitor, characterized by using as an index the degree of degradation or cleavage of a substrate by at least one enzyme selected from the group consisting of MSPL, MSPS, and TMPRSS13. An enzyme inhibitor obtained by the screening method according to claim 1. An antiviral agent containing the inhibitor or enzyme inhibitor according to claim 1 or 3 as an active ingredient in an effective amount. A virus infection and / or proliferation activator comprising an amount having a medicinal effect with at least one enzyme selected from the group consisting of MSPL, MSPS, and TMPRSS13 as an active ingredient.   Virus particles produced by using the activator according to claim 5 and / or components thereof. A therapeutic agent for a hormonal disease containing an effective amount of at least one enzyme selected from the group consisting of MSPL, MSPS and TMPRSS13 as an active ingredient. A therapeutic agent for hormonal diseases comprising an effective amount of an inhibitor for at least one enzyme selected from the enzyme group consisting of MSPL, MSPS, and TMPRSS13 as an active ingredient. A diagnostic agent for hormonal diseases using the gene expression level of at least one enzyme selected from the enzyme group consisting of MSPL, MSPS, and TMPRSS13 as an index.