WO2005056804A1 - Method of controlling function of receptor - Google Patents

Abstract

It is intended to provide a means of controlling or inhibiting the binding of a receptor occurring on cell surface to its agonist. Namely, a method of controlling the function of a receptor occurring on cell surface; a method of treating or preventing a disease the onset or pathological progress of which is caused by the function of a receptor or signal transduction mediated thereby and/or an infectious disease wherein the infection is caused by an interaction with a receptor; a remedy or a preventive for the above disease and/or the above infectious disease; utilization of a functional substance or the like for treating or preventing the above disease and/or the above infectious disease; utilization of a functional substance or the like for producing a drug for treating or preventing the above disease and/or the above infectious disease; and so on.

Description

 Specification

 Regulation of receptor function

 Technical field

 [0001] The present invention relates to a method for regulating the function of a receptor present on a cell surface, a disease in which the function of a receptor or the transmission of information via the receptor causes the onset or progression of symptoms, and the interaction with a Z or receptor. The present invention relates to a method for treating or preventing an infectious disease causing the disease, the use of the disease and Z or a functional substance for treating or preventing the infectious disease, and an agent for treating or preventing the disease and Z or the infectious disease.

 Background art

 [0002] Living organisms maintain their lives by the function of various components such as various organs, tissues, and cells. In order for the living body to respond to changes in internal and external conditions, control systems such as the nervous system and endocrine system need to regulate the functions between components and maintain coordination.

 [0003] Information transmission between cells is performed by neurotransmitters, cytokins, hormones and! /, A transmitter, and the information of the transmitter is transmitted into cells by specific receptors. Is transmitted. Some of the receptors have also been used as a foothold when pathogenic microorganisms and viruses enter cells.

 [0004] When it is necessary to adjust the information transmission function of a living body for a disease or other reasons, the function of the transmitter, the function of the receptor, or the function of both the transmitter and the receptor is adjusted. The goal can be achieved. For example, by coexisting a substance having a structure similar to that of a transmitter and capable of binding to the receptor of the transmitter, a substance that does not emit a signal from the receptor to the inside of the cell is allowed to coexist. Binding can be competitively inhibited. Such substances are called receptor antagonists.

[0005] The control of signal transduction using an antagonist is greatly affected by the abundance ratio between a transmitter and an antagonist and the affinity between an antagonist and a receptor. In addition, when the half-life of the antagonist in the living body is short, the The use of a second strike is not an effective measure.

 [0006] On the other hand, as a method for localizing a protein on a cell surface, a method using a signal peptide of tyrosine kinase 'receptor (for example, see Non-patent Document 1), a method using a GPI anchor signal sequence (for example, Patent Document 2), a method using the transmembrane domain of the PDGF receptor (eg, see Non-patent Document 3), a method using a mucin box rich in serine and threonine (eg, see Non-patent Document 4), glycophorin C Methods (for example, see Patent Document 1) have been developed.

 Patent Document 1: JP-A-2003-235577

 Non-patent literature l: Nature Biotechnology, vol. 15, pp. 1373-1377 (1997) Non-patent literature 2: FEBS Letters, vol. 458, pp. 299-303 (1999)

 Non-Patent Document 3: Gene Therapy, Vol. 8, pp. 1005-1010 (2001)

 Non-Patent Document 4: Biol. Chem., Vol. 278, pp. 10523-10530 (2003) Disclosure of the Invention

 Problems to be solved by the invention

[0007] In one aspect, the present invention provides a method for regulating or inhibiting the binding of a receptor present on a cell surface to its agonist, regulating, suppressing, or inhibiting the function of a receptor, and signaling transmission. Regulation, reduction or inhibition, treatment or prevention of a disease in which receptor function or the like causes the onset or progression of symptoms, delaying the onset or progression of a disease state in an individual suffering from the disease, etc. To provide a method for modulating the function of a receptor present on a cell surface, which enables the function of the receptor. In another aspect, the present invention provides a method for inhibiting the function of a receptor for an extracellular signal transduction substance such as a cytokin or a growth factor, and modulating, reducing or inhibiting signal transmission between cells by a signal transduction substance in a living body. To provide a method of modulating signal transduction in an animal that enables In another aspect, the present invention provides a method for treating or preventing a disease in which the function or the like of a receptor causes the onset or progression of symptoms, and treating or preventing an infectious disease in which interaction with the receptor causes infection. Receptor function or information transmission through it, which can prevent, delay the onset of disease, or progression of the disease in an individual suffering from the disease or infectious disease, causes onset or progression of symptoms. Disease and interaction with Z or receptor It relates to providing a method for treating or preventing an infectious disease, the action of which causes the infection. Further, in another aspect, the present invention relates to treating or preventing the disease, treating or preventing the infectious disease, delaying the onset of the disease or progression of the disease in an individual suffering from the disease or the infectious disease. To provide a therapeutic or preventive agent for the disease and Z or the infectious disease, which is capable of causing a disease or the like. In still another aspect, the present invention relates to treating or preventing the above-mentioned disease, treating or preventing the above-mentioned infectious disease, delaying the onset or progression of a disease state in an individual suffering from the above-mentioned disease or infectious disease. The use of a functional substance for the treatment or prevention of said disease and Z or said infectious disease. Further, according to another aspect of the present invention, a medicament capable of treating or preventing the above-mentioned disease, the above-mentioned infectious disease and the like, and delaying the onset and progress of the disease state in an individual suffering from the above-mentioned disease or infectious disease is produced. The use of the functional substance or the nucleic acid for the manufacture of a medicament for the treatment or prevention of the disease and z or infectious disease, and the like. Another object of the present invention is also clear in the description power of the present specification. Means for solving the problem

 [0008] The present invention localizes a polypeptide that is not naturally localized on the cell membrane surface to the cell membrane surface, and causes an interaction between a receptor present on the cell surface and the polypeptide. Thus, it relates to providing a means for modulating or inhibiting the binding of the receptor to its agonist.

 [0009] In a first aspect, the present invention provides a functional substance containing a region of a polypeptide localized on the cell membrane surface that contributes to localization of the membrane and a substance that can interact with a receptor on the cell surface. And a method for regulating the function of a receptor present on the cell surface, which comprises localizing the receptor on the cell membrane surface.

 [0010] As one embodiment of the method for regulating the function of the receptor, a method for inhibiting the interaction between a receptor present on the cell surface and an extracellular substance is exemplified. Examples of the “polypeptide localized on the cell membrane surface” include a polypeptide containing a mucin box.

[0011] The "substance capable of interacting with a cell surface receptor" may be a peptide or a polypeptide. In this case, the substance may be a polypeptide or a polypeptide localized on the cell membrane surface. It can be introduced into a cell as a nucleic acid encoding a fusion polypeptide of a region contributing to membrane localization and a polypeptide capable of interacting with a cell surface receptor. In addition, a method for introducing a gene encoding the fusion polypeptide, that is, a nucleic acid, into a cell is exemplified as a means at that time.

 [0012] The "substance capable of interacting with a cell surface receptor" includes growth factors, cytokines, chemokines, peptide hormones, polypeptide hormones, cell adhesion factors, toxins, antibodies, microorganism-derived proteins, viruses, and the like. Derived proteins (eg, proteins of the outer membrane of the virus), fragments thereof, and the like.

 [0013] In a second aspect, the present invention provides a functional material containing a region that contributes to localization of a polypeptide localized on the cell membrane surface and a substance that can interact with a cell surface receptor. Is localized on the cell membrane surface.

 According to a third aspect of the present invention, there is provided a functional substance comprising a region contributing to localization of a membrane of a polypeptide localized on a cell membrane surface and a substance capable of interacting with a cell surface receptor. Is localized on the cell membrane surface of an individual at the site of a disease or a site where a disease is likely to develop, and the function of the receptor or the transmission of information through it may cause the onset or progression of symptoms. The present invention relates to a method for treating or preventing a disease and an infection in which interaction with Z or a receptor causes infection.

 [0015] In a fourth aspect, the present invention provides a functional substance containing a region of a polypeptide localized on the cell membrane surface that contributes to membrane localization and a substance capable of interacting with a cell surface receptor. Treatment or prevention of diseases in which the function of the receptor or signal transmission through it causes onset or progression of symptoms, and in which the interaction with Z or the receptor causes infection, comprising as an active ingredient Agent.

[0016] In a fifth aspect, the present invention provides a method for treating diseases in which the function of a receptor or information transmission therethrough causes onset or progression of symptoms, and infectious diseases in which interaction with Z or a receptor causes infection. For treatment or prevention, a functional substance containing a region of a polypeptide localized on the cell membrane surface contributing to membrane localization and a substance capable of interacting with a cell surface receptor, or the functional substance is coded. To the use of nucleic acids. [0017] In a sixth aspect, the present invention provides a method for treating diseases in which the function of a receptor or information transmission therethrough causes onset or progression of symptoms, and infectious diseases in which interaction with Z or a receptor causes infection. A functional substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a receptor on the cell surface for the manufacture of a medicament for treatment or prevention. Alternatively, the present invention relates to the use of a nucleic acid encoding the functional substance.

 The invention's effect

According to the method for regulating the function of a receptor present on a cell surface of the present invention, it is possible to regulate, reduce, or inhibit the binding of a receptor present on a cell surface to its agonist, the function of the receptor, and signal transduction. In addition, the present invention exerts an excellent effect of delaying the onset and progress of a disease state in an individual suffering from the disease. Further, according to the method for regulating the function of a receptor of the present invention, an excellent effect is exhibited in that a disease in which the function of the receptor or the like causes the onset or progression of symptoms can be treated or prevented. ADVANTAGE OF THE INVENTION According to the method of regulating signal transduction in animals of the present invention, it is possible to inhibit the function of receptors for extracellular signal transduction substances such as cytokins and growth factors, and signal between cells by the signal transduction substance in vivo. It has an excellent effect of regulating, reducing and inhibiting transmission. Further, according to the treatment or prevention method of the present invention, it is possible to treat or prevent a disease in which the function or the like of a receptor causes the onset or progression of symptoms, an infectious disease in which interaction with the receptor causes an infection, or the like. The present invention exerts an excellent effect of delaying the onset and progress of a disease state in an individual suffering from the disease or infectious disease. Furthermore, according to the therapeutic or prophylactic agent of the present invention, the disease, the infectious disease, etc. can be treated or prevented without repeatedly administering drugs, etc. The present invention has an excellent effect of delaying the onset and progress of a disease state in an affected individual. According to the use of the functional substance or the nucleic acid encoding the functional substance for the treatment or prevention of the disease and Z or the infectious disease of the present invention, the disease, the infectious disease and the like are repeated by a drug and the like. It is possible to treat or prevent the disease without administration and to delay the onset and progress of the disease state in an individual suffering from the above-mentioned disease or infectious disease. Preparation of a medicament for treating or preventing the aforementioned diseases and Z or infectious diseases of the present invention. According to the use of the functional substance or the nucleic acid for production, the disease, the infectious disease, and the like can be treated or prevented, and the activity and the selectivity are excellent. An excellent effect of producing a drug capable of delaying the onset and progression of the disease state in an affected individual can be produced. According to the present invention, a polypeptide which is not naturally localized on the cell membrane surface is localized on the cell membrane surface to cause an interaction between a receptor present on the cell surface and the polypeptide. Thus, a method for regulating or inhibiting the binding between the receptor and its agonist is provided.

 Brief Description of Drawings

 FIG. 1 is a graph showing the ability of a CD4-positive cell line to infect gpl20-rsGFP virus.

 The horizontal axis is the number of viruses used for virus infection, and the vertical axis is the rsGFP expression positive rate in cells after virus infection. In FIG. 1, a black circle indicates a CD4-positive cell line [CD4-expressing He La cells (HeLaZCD4)], and a black triangle indicates a CD4-positive cell line (MOLT-3).

 FIG. 2 is a diagram showing a comparison of gpl20-rsGFP virus infectivity to CD4 negative cell lines (HeLa cells) and CD4 positive cell lines (HeLaZCD4 cells). The horizontal axis is the added cell line, and the vertical axis is the gpl20 expression positive rate in the cells after virus infection.

 FIG. 3 is a diagram showing gp120 expression on the cell surface by mucin box gp120 gene transfer. The horizontal axis is the name of the plasmid introduced into the HeLa cell, I is pTriEx3-NEO, II is pcDNAkCD, III is pTriEx3-gpl20, and IV is pCA-SMgpl20. The vertical axis is the rsGFP expression positive rate in cells after each plasmid introduction

FIG. 4 is a diagram showing the detection of cell surface CD4 in cells into which mucin box gpl20 gene has been introduced. The horizontal axis is the name of the plasmid introduced into HeLaZCD4 cells. I is pTriEx3-NEO, II is pcDNAkCD, III is pTriEx3-gpl20, and IV is pCA-SMgpl20. The vertical axis represents the CD4 expression positive rate in the cells after each plasmid introduction.

FIG. 5 is a diagram showing the inhibition of gp120-rsGFP virus infection by transduction of the mucin box gp120 gene. The horizontal axis is the name of the plasmid introduced into HeLaZCD4 cells. , I is pTriEx3-NEO, II is pcDNAkCD, III is pCA-SMgpl20. The vertical axis indicates the positive rate of rsGFP expression in cells infected with gpl20-rsGFP virus after introduction of each plasmid.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0024] In one aspect, the present invention provides a functional substance containing a region that contributes to localization of a polypeptide localized on the cell membrane surface and a substance that can interact with a cell surface receptor. And a method for regulating the function of a receptor present on the cell surface, characterized by localizing the receptor on the cell membrane surface.

 [0025] The present invention provides a method for introducing into a cell a functional substance constructed by binding a substance capable of interacting with a receptor to a region of a polypeptide localized on the cell membrane surface which contributes to localization of the membrane. On the basis of the surprising finding of the present inventors that a substance capable of interacting with the receptor is localized on the surface of the cell, whereby the function of the receptor on the cell surface can be regulated.

 [0026] The method for regulating receptor function of the present invention provides a functional substance comprising a region contributing to localization of a membrane of a polypeptide localized on a cell membrane surface and a substance capable of interacting with a cell surface receptor. There is one major feature that is used. Therefore, according to the method of regulating the receptor function of the present invention, it is possible to selectively regulate the function of the receptor of the target cell, and it is also excellent that the function of the receptor is continuously regulated. It is effective.

[0027] Further, the method for regulating a receptor function of the present invention has one significant feature in that the functional substance is localized on the surface of a cell membrane to regulate the function of a receptor present on the cell surface. Therefore, according to the method of regulating the receptor function of the present invention, for example, a polypeptide that is not naturally localized on the cell membrane surface is localized on the cell membrane surface, and the receptor present on the cell surface and the polypeptide The interaction between the receptor and the receptor can regulate or inhibit the binding between the receptor and the agonist. In addition, since a substance capable of interacting with a cell surface receptor is localized on the cell surface, the effect may be substantially lost due to diffusion as in a conventional control method using a soluble substance. Demonstrates the superior effect of enabling continuous receptor control To do. Further, in the method of regulating the receptor function of the present invention, by selecting a method having high specificity for a target cell as a method for introducing into a cell, the method has substantially no effect on receptors of other cells. When it becomes possible to control cell-specific receptor functions!す る Excellent effect.

 [0028] According to the method for regulating a receptor function of the present invention, the interaction between a receptor present on the cell surface and an extracellular substance can be inhibited.

 [0029] The "polypeptide localized on the cell membrane surface" used in the method for regulating a receptor function of the present invention is not particularly limited. Examples of the "polypeptide localized on the cell membrane surface" include, for example, a polypeptide having an amino acid sequence called "mucin box" rich in serine, threonine and proline (ceramidase; sucrase isomaltase i. Biol. Chem., Vol. 275, pp. 6566-6572 (2000)]; Dipeptinoleveptidase IV [Exp. Cell Res., Vol. 258, pp. 184-194 (2000)]; Biol. Chem., Vol. 273, pp. 30263-30270 (1998)]; Rat bile salt-dependent 'linose i. Biol. Chem., Vol. 272, pp. 27353-27361 (1997)]; Human MUC2 Q. Biol. Chem., Vol. 269, pp. 2440-2446 (1994)], polypeptides having transmembrane domains [site force receptors (EGF receptor, PDGF receptor, FGF receptor, etc.) ), Various hormone receptors], GPI anchor polypeptide [Alkaline phosphatase, VSG of Trypanosoma parasites [Science, vol. 216, p. 696 (1982)], CD55 or CD59 F. Biochem. Of erythrocyte membrane, vol. 104, p. 633 (1988)], Thy- l [Nature, Vol. 333, pp. 269-272 (1988)] and the like. Among them, a mucin box, which is a polypeptide having a relatively small size, is preferable from the viewpoint of use as a fusion polypeptide.

[0030] In the method of regulating the receptor function of the present invention, the "region involved in localization to the cell membrane" in the "polypeptide localized on the cell membrane surface" is not particularly limited. Can be used. In the method of regulating the receptor function of the present invention, the use of the "region involved in localization to the cell membrane" is advantageous in that the size of the fusion polypeptide expressed in cells can be reduced. Examples of the “region involved in localization to cell membrane” include, for example, mucin box, signal peptide, transmembrane domain, GP I anchor signal and the like. Hereinafter, the region is sometimes referred to as a “membrane localized region”.

 [0031] In the method for regulating a receptor function of the present invention, the "polypeptide localized on the cell membrane surface" is in a range that does not lose the ability to localize to the cell membrane, that is, in a range that sufficiently exerts the ability. Or one or more amino acids thereof, that is, at least one, specifically, one or more, more specifically, one or several amino acids (i.e., amino acid residues) At least one of substitution, deletion, insertion or addition of a polypeptide, i.e., one or more, specifically, one or several introduced polypeptides (a variant of a polypeptide localized on the cell membrane surface). ) Can also be used. The noriant of the polypeptide localized on the cell membrane surface may be a naturally-occurring noriant, a polypeptide into which amino acid residue substitution, deletion, insertion, or addition has been artificially introduced. It may be.

 [0032] In the present specification, the "part of the polypeptide localized on the cell membrane surface" is synonymous with the polypeptide of the "region involved in localization to the cell membrane".

 [0033] The ability to localize to the cell membrane can be determined, for example, by using a fusion polypeptide in which a detectable marker polypeptide is arranged downstream of the polypeptide to be evaluated (the polypeptide to be evaluated and the marker polypeptide are different from each other). The fusion nucleic acid encoding the fusion polypeptide can be evaluated by introducing the nucleic acid into a suitable cell and expressing it. Here, when the fusion polypeptide is present on the cell surface, the polypeptide to be evaluated is `` a polypeptide normal located on the cell membrane surface '' having the ability to localize on the cell membrane. Is an indicator of The presence of the fusion polypeptide on the cell surface can be confirmed using, for example, the function of the marker polypeptide itself (eg, enzyme activity, fluorescence, etc.) or an antibody against the marker polypeptide.

 [0034] In the method for regulating the receptor function of the present invention, for example, a region containing a mucin box can be extracted from the mucin box-containing polypeptide and used.

[0035] The mucin box includes, for example, a mucin box having the amino acid sequence shown in SEQ ID NO: 1. The mucin box having the amino acid sequence represented by SEQ ID NO: 1 was obtained from the N-terminal part of rat kidney-derived ceramidase [Journal of Neurological Chemistry, vol. 276, pp. 26249-26259 (2001)]. Mucin found It is a box.

In addition, the mucin box includes amino acid numbers: 10, 14, 15, 16, 18, 19, 21, 22, 24, 26, 27, 29 in the amino acid sequence represented by SEQ ID NO: 1. 0—the site to which a linked sugar chain can be added "exists. The amino acid residue in the “site to which an o-linked sugar chain can be added” is not particularly limited as long as it can add an O-linked sugar chain. For example, threonine (T) Or serine (S) is preferred. The mucin box that can be used as a membrane localization region in the present invention includes at least 40%, preferably at least 50%, more preferably at least 60%, and more than the amino acid sequence shown in SEQ ID NO: 1. Preferably, those having at least 70% sequence identity and having the ability to localize to the cell membrane are mentioned. Here, the sequence identity is a value calculated and adjusted appropriately by the BLAST algorithm under the conditions of expect value 10, wordsize 5, Cost to open gap 丄 丄, and ost to extend gap 1. Means Specifically, the mucin box may be, for example, the substitution of the amino acid residue at position 1 with T, the substitution of the amino acid residue at position 2 with T, the amino acid at position 3 in SEQ ID NO: 1, Substitution of a residue with P or T, substitution of amino acid residue at position 4 with T, substitution of amino acid residue at position 5 with Q, substitution of amino acid residue at position 6 with A , Substitution of amino acid residue at position 7 with F or P, substitution of amino acid residue at position 8 with T, substitution of amino acid residue at position 9 with P, substitution of amino acid residue at position 11 Substitution of amino acid residue at position 12 with N, S or A, substitution of amino acid residue at position 13 with S, substitution of amino acid residue at position 15 with P Substitution, substitution of amino acid residue at position 17 with S or P, substitution of amino acid residue at position 18 with P, T or A, substitution of amino acid residue at position 19 with P, position 20 Substitution of an amino acid residue of Substitution of amino acid residue at position 21 with S or I, substitution of amino acid residue at position 23 with S or T, substitution of amino acid residue at position 24 with P or A, amino acid at position 25 Substitution of a T for a residue, substitution of an amino acid residue at position 26 for I, substitution of an amino acid residue for position 27 with S, substitution of an amino acid residue for position 28 with S, position 29 Amino acid residue at position T, amino acid residue at position 30 with T or Q, amino acid residue at position 31 with N, S, T or A, amino acid at position 32 Substitution of residue F or J, substitution of amino acid residue at position 33 with R, substitution of amino acid residue at position 34 with T, substitution of amino acid residue at position 35 with E or T Substitution of the amino acid residue at position 36 with T, At least the deletion of the sequence with the 1st position, the 8th position of the amino acid, the deletion of the 1st position, the deletion of the 7th position, the deletion of the sequence with both the 32nd position and the 36th position, etc. One may have a range capable of localizing to the cell membrane.

 [0037] The mucin box having the amino acid sequence of SEQ ID NO: 1 is a polypeptide consisting of 36 amino acid residues, and localizes a heterologous protein (ie, a heterologous polypeptide, a peptide, etc.) to the cell membrane surface. It is very useful for The mucin box can be used in the present invention, for example, by linking to a signal peptide. Here, the signal peptide that can be used includes, for example, a signal peptide derived from a secretory enzyme, a signal peptide derived from cytodynamics, and a signal peptide derived from chemokines, as long as the peptide is effective for passage through the cell membrane of the target cell. Signal peptide and the like.

 [0038] The "substance that interacts with a cell surface receptor" is not particularly limited, but is preferably a peptide or a polypeptide because it can be a fusion polypeptide with the membrane-localizing region. It is. In addition, substances other than peptides and polypeptides can be used in the present invention as long as they can be bound to polypeptides localized on the cell membrane surface by a known method. Here, in the present specification, the polypeptide refers to a polypeptide composed of 11 or more amino acid residues. In addition, a peptide refers to a peptide composed of 210 amino acid residues. The term "interacts with the receptor" means that binding to the receptor, association and competition with the agonist for Z or binding to the receptor occur.

 [0039] The "substance that interacts with a cell surface receptor" is not particularly limited, but is preferably a substance that does not transmit an intracellular signal such as that induced by binding to the receptor. . Therefore, in the method of regulating the receptor function of the present invention, an antagonist of the receptor, an antibody recognizing the receptor and the like can be used.

[0040] Examples of the "antagonist of receptor" include analogs of receptor agonists, mutants of agonists, portions of agonists (eg, binding domains with receptors), and the like. Is mentioned. Examples of the agonist variants include, when the agonist is a peptide or a polypeptide, those having mutations such as amino acid substitution, deletion, insertion, etc., those having a modified amino acid side chain, agonists Modified sugar chains present in No. The “antibody that recognizes the receptor” also includes, for example, antibody fragments, single-chain antibodies, and the like. Examples of the “antibody fragment” include a Fab fragment obtained by digesting a monoclonal antibody with nopain, and an F (ab ′) fragment obtained by digesting a monoclonal antibody with pepsin, for example. Also includes

 2

 I do. Further, in the present specification, “an antibody that recognizes a receptor” has the same meaning as an antibody that binds to the receptor.

[0041] The purpose is not to control signal transduction from a receptor! / In some cases, for example, when the purpose is to control the entry of a pathogen into a cell via a receptor or the control of endocytosis, Ghosts and fragments thereof (portions that can interact with the receptor) can be used in the present invention.

 [0042] As used herein, the term "substance that interacts with the receptor" includes a growth factor [fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), nerve growth factor ( NGF), cytokins (interferons, interleukins, TNF, etc.), chemokines (CXC chemokines, CC chemokines, etc.), peptide hormones (LH-RH, angiotensin, etc.), polypeptide hormones (growth hormone, Human chorionic gonadotropin (HCG), etc.], cell adhesion factors (fibronectin, thrombosbondin, etc.), toxins (cholera toxin, etc.), components (polysaccharides, proteins, etc.) present in the cell wall of microorganisms and the outer membrane of viruses, etc. An example is shown. Furthermore, antibodies or derivatives thereof that recognize the receptor as described above can also be used in the present invention.

 Regarding the functional substance, the method of binding the membrane localization region (polypeptide) to a substance that interacts with a receptor on the cell surface is not particularly limited, and the membrane localization region (polypeptide) is not particularly limited. An appropriate method may be selected depending on the characteristics of both the peptide and the substance that interacts with the cell surface receptor. Also, the two may be directly connected or may be connected via an appropriate linker.

As used herein, the term “functional substance” refers to a first moiety (moiety) that contributes to localization of a membrane of a polypeptide localized on a cell membrane surface. And a second part (moiety) capable of interacting with a receptor on the cell surface, wherein the first part and the second part are fused or complexed! / You can! [0045] When the substance that interacts with the receptor is a peptide or polypeptide, it can be a fusion polypeptide with a membrane-localized region (polypeptide). The fusion peptide is, for example, a membrane-localizing region (polypeptide), a membrane-localizing region (polypeptide) that can be a polypeptide capable of acting only with a peptide or polypeptide that interacts with the receptor, and a receptor. It may be a polypeptide in which another appropriate peptide or polypeptide is inserted as a linker between the acting peptide or polypeptide and the like. Here, the substance that interacts with the receptor includes a so-called noriant as long as it exerts a function of regulating and inhibiting the binding between the receptor and the agonist. The noriant includes an amino acid residue (hereinafter, also referred to as a similar amino acid residue in the present specification) having a function similar to hydrophobicity, charge, pK, and three-dimensional structure. Substitution, which can change the steric structure and fold structure of the polypeptide only to maintain the original receptor function! 維持 Variants with conservative substitutions such as substitution with amino acid residues, ΝTerminal or C-terminal And variants lacking the same. Here, similar amino acid residues are, for example, amino acid residue group 1: glycine and alanine; amino acid residue group 2: palin, isoleucine, and leucine; amino acid residue group 3: aspartic acid, glutamic acid, asparagine and glutamine; Amino acid residue group 4: serine, threonine; amino acid residue group 5: lysine, arginine; amino acid residue group 6: means the relationship between amino acids belonging to phenylalanine and tyrosine.

When the substance that interacts with the receptor is a peptide or polypeptide, it is possible to use a nucleic acid that encodes a membrane-localizing region (polypeptide) and a substance that interacts with the receptor. it can. In this case, the nucleic acid encoding the localization region (polypeptide) is one in which the encoded polypeptide has the ability to localize the polypeptide to the cell membrane. The nucleic acid encoding the localized region (polypeptide) may be, for example, a stringent condition, preferably a highly stringent condition, with the nucleic acid of the antisense strand of the nucleic acid encoding the amino acid sequence of SEQ ID NO: 1. It may be a nucleic acid that hybridizes to a nucleic acid. Here, as the nucleic acid that hybridizes with the nucleic acid of the antisense strand under stringent conditions, more specifically, for example, 6 × SSC (1 × SSC composition: 0.15 M NaCl, 0.015 M sodium, pH 7. 0) and 0.5 and 5 weight _^0/0 SDS and 5 X Den'noヽNoreto lOO ^ g / ml Denatured salmon sperm DNA and 50% by volume formamide in a solution at room temperature, more stringent at 42 ° C, more stringent at 60 ° C for 10 hours, low ion Strength, e.g., 2 X SSC, more stringent, 0.1 X SSC, etc. and Z or higher temperature, room temperature, 42 ° C or higher, more stringent, 55 ° C, more stringent Examples thereof include nucleic acids and the like that can detect the formation of a hybrid with the nucleic acid of the antisense strand when the hybridization is performed under conditions of washing at 60 ° C. or the like. Such a nucleic acid has at least 50%, preferably 60% or more, more preferably 70% or more sequence identity with the base sequence of the nucleic acid encoding the amino acid sequence of SEQ ID NO: 1, and is encoded. A polypeptide is a nucleic acid that has the ability to localize the polypeptide to the cell membrane. A nucleic acid encoding a substance that interacts with the receptor has the physiological activity of the corresponding natural substance and hybridizes under the above-mentioned conditions with the nucleic acid of the antisense strand of the nucleic acid encoding the amino acid sequence of the substance. The nucleic acid may be used.

 [0047] The function of the substance capable of interacting with the receptor on the cell surface is determined by detecting the formation of a bond between the substance and the corresponding receptor, for example, by surface plasmon analysis, yeast two-hybrid method, or the like. Can be evaluated. For example, in the case of surface plasmon analysis, a sensor chip on which a receptor is immobilized and a solution containing a substance that can interact with the receptor on the cell surface are sent at a constant flow rate, and appropriate detection means (for example, optical Detection (fluorescence, fluorescence polarization, etc.), combined with a mass spectrometer (matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer: MALDI-TOF MS, electrospray ionization mass spectrometer: ESI) -MS etc.)], the formation of the bond can be measured by detecting the interaction between the above-mentioned factor and the test substance as optical fluctuation or mass fluctuation. In the surface plasmon analysis, for example, when a change is caused by the introduction of a substance capable of interacting with the receptor on the cell surface by optical sensorgram or mass sensorgram transfer, it becomes an indicator of the formation of binding. Indicates that the substance interacts with the receptor.

[0048] A functional substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a cell surface receptor can be targeted by an appropriate method. It is introduced into cells, ie, cells where modulation of the receptor is desired. Examples of the introduction method include known methods such as a ribosome method, a perforation method, and a method using a virus vector.

 [0049] In one embodiment of the present invention, a gene encoding a fusion polypeptide of a membrane-localized region and a peptide or polypeptide that interacts with a receptor, ie, a nucleic acid, is constructed and placed in a target cell. By introducing and expressing the fusion polypeptide, the polypeptide interacting with the receptor can be localized on the surface of the target cell. The positional relationship between the membrane-localizing region and the peptide or polypeptide that interacts with the receptor in the fusion polypeptide is not particularly limited, and the membrane-localizing region to be used, the type of peptide or polypeptide used, etc. The polypeptide may be fused in an appropriate order according to the conditions. For example, the above mucin box is usually added to the C-terminal side in the case of a force GPI anchor signal added to the N-terminal side of a peptide or polypeptide that interacts with the receptor.

 [0050] Further, the fusion polypeptide has a peptide other than these elements, such as a secretory signal, as long as its function, that is, localization on the cell surface and interaction with the receptor are not impaired. May be. Further, a fusion polypeptide in which a linker polypeptide is inserted between a membrane-localizing region and a peptide or polypeptide that interacts with the receptor can also be used in the present invention.

 [0051] The gene encoding the fusion polypeptide, ie, the nucleic acid, may be expressed by an appropriate promoter operable in a target cell. For example, operably positioned downstream of a promoter that specifically promotes transcription depending on the type of cell, it is possible to specifically express in a desired target cell and regulate the function of the receptor. is there. When a promoter that promotes transcription in accordance with a specific inducer is used, the expression of the fusion polypeptide can be artificially controlled using the inducer.

[0052] The method for introducing the gene encoding the fusion polypeptide, ie, the nucleic acid, is not particularly limited, but a method using a non-viral vector such as a calcium phosphate method, a lipofection method, or a ribosome method; Virus vector (Lentivirus vector And adenovirus vectors, adeno-associated virus vectors, and methods using viral vectors such as vaccinia virus vector. When it is desired to continuously express the fusion polypeptide in target cells, the use of a retrovirus vector is preferred.

[0053] According to the method for regulating the function of a receptor of the present invention, an interaction occurs between a substance that interacts with the receptor and the receptor, thereby suppressing or inhibiting the function of the receptor. Therefore, by using the method of the present invention to inhibit the function of a receptor for an extracellular signal transmitting substance typified by a growth factor or a site force-in, the intercellular transmission of the signal transmitting substance in a living body can be prevented. Signaling can be reduced or blocked. That is, as one of the embodiments of the present invention, a functional substance containing a region contributing to localization of a membrane of a polypeptide localized on a cell membrane surface and a substance capable of interacting with a receptor on a cell surface, A method for regulating signal transduction in an animal (for example, a non-human animal), which is characterized in that the signal transduction is localized on the cell membrane surface. In particular, the method of the present invention is useful for treating or preventing a disease caused by an excessive signal of an information substance binding to a receptor. The term “treatment” as used in the present invention also includes a mode of delaying the onset and progress of a disease state in an individual suffering from the disease, infectious disease or the like.

 [0054] Tumor cells produce endothelial cell growth factor called vascular endothelial cell growth factor (VEGF) and induce angiogenesis around the tumor tissue. By inhibiting the function of the VEGF receptor in endothelial cells, angiogenesis can be suppressed and tumor growth can be suppressed.

 [0055] CD28 is known as an auxiliary signal for antigen recognition in T cells. Since this accessory signal is also involved in autoimmune diseases and graft-versus-host disease (GVHD), for example, using the method of the present invention, CD28 of donor-derived T cells transferred to patients after hematopoietic stem cell transplantation can be used. GVH during donor lymphocyte transfer (DLI) by inhibiting function

D can be suppressed.

When a receptor used when a pathogen such as a microorganism or a virus enters a cell is blocked by the method of the present invention, it is possible to protect the cell from infection with the microorganism or virus. Delays onset and pathology in individuals infected with the pathogen Is possible.

 [0057] As described in the Examples below, infection with the human immunodeficiency virus (HIV) can be controlled by using the method of the present invention.

 [0058] Since HIV recognizes the CD4 receptor molecule and infects cells, the viral outer membrane protein (gpl20 molecule) that binds to CD4 in cell recognition by the HIV is transferred onto the surface of CD4-positive cells by the method of the present invention. In this case, infection with a retrovirus (a pseudotyped virus) having gp120 as an envelope is inhibited.

 [0059] In the case of HIV, chemokine receptors are recognized in addition to the CD4, and therefore, the same effect can be obtained by regulating the function of the receptor by the method of the present invention.

 [0060] Epstein-Barr virus, CD21, rhinovirus, ICAM-1, rabies virus, acetylcholine receptor, vaccinia virus, EGF receptor, and influenza virus, sialic acid on the cell surface. Recognize and infect each. Therefore, the functions of these receptors and the like can be regulated by the method of the present invention to suppress or prevent infection of these viruses.

 [0061] Furthermore, it is known that when a virus germinates from an infected cell, the virus expresses a protein expressed on the surface of the infected cell on the surface of the virus cell Q [. Virol., Vol. 9329-9336 (1998)]. According to the method of the present invention, a part of the env-derived protein (virus outer membrane protein) necessary for virus adsorption and invasion into cells as a fusion polypeptide with a region contributing to membrane localization on the cell surface. When expressed, the env-derived protein that cannot perform its function during invasion is retained on the surface of the virus that germinates from the cell. Thus, it is possible to secondarily suppress viral infection of cells, prevent or delay the onset of various symptoms caused by the infection, and delay the progression of the disease state. For example, by using the method of the present invention to express a fusion protein of gP120 and a membrane localization region on the cell membrane targeted for HIV infection, this cell force is also expressed on the HIV surface when HIV germinates. It is possible to express env-derived proteins that have no invasion function.

[0062] The method of the present invention is not limited to the method related to virus infection. example For example, the binding of the toxin produced by the bacteria to the receptor can be controlled by using the method of the present invention.

 [0063] Since the toxin produced by hemorrhagic Escherichia coli or the like binds to a glycolipid receptor called Gb3 on the target cell, an antibody against the glycolipid Gb3 is applied to the cell surface of the target cell by the method of the present invention. Expression of the light chain inhibits the binding of verotoxin to the glycolipid receptor.

 [0064] Furthermore, among the sugar chain receptors recognized by microorganisms (for example, protein nucleic acid enzyme, Vol. 37, pi 810-1819 (1992)), the functions of sugar chain receptors suitable for the target microorganisms and the like are described. By adjusting by the method of the present invention, bacteria (for example, cholera (G), tetanus (G)

 Ml

 ), Shigella (G Cer), Vibrio parahaemolyticus (G), etc .: Parentheses indicate sugar chain receptors], protozoa

Dlb b3 Tib

 In addition, virus infection can be suppressed.

 [0065] In another aspect, the present invention provides a functional substance containing a region contributing to localization of a membrane of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a cell surface receptor. The function of the receptor or the transmission of information through it, or the cause of the progression of symptoms, characterized in that it is localized on the cell membrane surface at the site of a disease or a site where the disease may occur in the individual. The present invention relates to a method for treating or preventing an infectious disease in which an interaction with Z or a receptor causes infection.

 According to the method of treatment or prevention of the present invention, a functional substance containing a region of a polypeptide localized on the cell membrane surface that contributes to localization of the membrane and a substance that can interact with a receptor on the cell surface Is localized on the surface of the cell membrane at the site of the disease in the individual or at the site where the disease is likely to occur, so that the diffusion of a soluble substance continuously prevents the effect from being substantially lost. Therapeutic or prophylactic effect of a disease in which a receptor function or the like causes the onset or progression of symptoms, or an infectious disease or the like in which interaction with the receptor causes an infection at a disease site or a site where a disease is likely to develop. Can exert an excellent effect.

 [0067] The treatment or prevention method of the present invention can be performed, for example, in the same manner as the method for regulating the function of the receptor.

[0068] The above-mentioned "receptor function or information transmission via the receptor may cause the onset or progression of symptoms. The "causal disease" includes, for example, tumor, leukemia, autoimmune disease, GVHD and the like.

 [0069] The "infectious diseases caused by interaction with the receptor causing infection" are caused by infectious diseases caused by microorganisms and viruses (eg, human immunodeficiency virus, human hepatitis virus, etc.) and viral infection. Carcinogenesis, immunodeficiency and the like.

 [0070] The therapeutic or prophylactic effect of the therapeutic or prophylactic method of the present invention can be measured, for example, by examining the response of the receptor to agonist in a target cell in which a substance that interacts with the receptor is localized on the cell surface. And susceptibility to a pathogen infecting via the receptor. Here, a decrease or loss of agonist reactivity or susceptibility to a pathogen is an indicator that the therapeutic or preventive effect of the treatment or prevention method of the present invention has been obtained.

 [0071] According to the treatment or prevention method of the present invention, a substance that interacts with the receptor is localized on the surface of the target cell. As a result, a therapeutic or preventive effect can be obtained effectively without substantially losing the effect. In addition, by adopting an introduction method specific to a target cell, the expression of the therapeutic or preventive effect can be more locally concentrated.

 [0072] In one embodiment of the present invention, a functional group containing a region that contributes to localization of a membrane of a polypeptide localized on the cell membrane surface and a substance that can interact with a cell surface receptor is used. Modifiers of receptor function containing a substance as an active ingredient are exemplified. As the regulator, functional substances used in the method for regulating the receptor function of the present invention described above can be used. Further, the active ingredient may be a gene encoding the functional substance, that is, a nucleic acid containing a nucleic acid.

 [0073] The modulator of the receptor function described above can be mixed with a pharmaceutically acceptable carrier, if necessary, to form an arbitrary preparation. The dosage form of the preparation is not particularly limited, and a dosage form suitable for a cell in which the function of the receptor is required and a tissue or organ containing the cell may be selected.

[0074] The nucleic acid encoding the functional substance may be a disease in which the function of the receptor or the transmission of information through the function causes onset or progress of symptoms, and interaction with Z or the receptor. Can be used as an agent for treating or preventing a disease that can cause infection. Examples of the drug include a drug containing a fragment containing the nucleic acid encoding the functional substance, a vector into which the nucleic acid is inserted, a virus vector into which the nucleic acid is inserted, and the like as an active ingredient.

 [0075] As an active ingredient, a drug containing a fragment containing a nucleic acid encoding the functional substance or a vector into which the nucleic acid has been inserted can be used to prepare cultured cells or live cells in the form of an appropriate solution containing the active ingredient. It can be used for introduction into cells in the body. Examples of the solution include solutions composed of pharmacologically acceptable components, for example, distilled water for injection, physiological saline, and phosphate buffered physiological saline. The solution may contain stabilizers and other pharmacologically acceptable additives. In addition, the dosage form should be such that it contains known components for gene transfer into cells, such as ribosomes, cationic lipids, calcium phosphate, etc., or is mixed with these components at the time of use. You can also.

 [0076] Drugs containing virus particles as an active ingredient are not particularly limited!を is a known vector commonly used in the gene transfer method, such as a retrovirus vector (including a lentivirus vector), an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a herpes virus vector, and the like. It can be manufactured using. Drugs containing these virus vectors as an active ingredient, that is, drugs containing virus particles containing the virus vector as an active ingredient, can be obtained by formulating in the form of a solution containing the virus particles. . Examples of the solution include physiological saline, phosphate buffered saline, and the like, and such a solution may further contain a stabilizer or other pharmacologically acceptable additives. Yo,

[0077] Furthermore, cells into which the nucleic acid encoding the functional substance has been introduced may also be a disease in which the function of the receptor or the signal transduction interpreting the function causes onset or progression of symptoms, and interaction with Z or the receptor. Can be used as a drug for treating or preventing a disease that may cause infection. For example, by introducing a nucleic acid encoding the above-described functional substance into a cell collected from a living body by a known method, a cell in which the function of a cell surface receptor is regulated can be produced. Since the action of an agonist on the receptor is regulated, the cell functions as a receptor or information to interpret the function. It has the effect of treating or preventing diseases whose signaling causes onset or progression of symptoms, and diseases whose interaction with Z or receptors may cause infection. For example, cells in which the function of a receptor associated with the infection of a pathogen is regulated have reduced susceptibility to the pathogen, and thus are effective in preventing infection.

 [0078] The drug containing the above-mentioned cells as an active ingredient can be obtained by formulating according to a known cell-containing preparation.

 [0079] The agent for regulating a receptor function of the present invention can be used for treatment and prevention of diseases requiring regulation of the function of the receptor (eg, neoplastic disease, autoimmune disease, GVHD, etc.) Useful for prevention.

 [0080] Further, as one embodiment of a powerful regulator, a function comprising a region contributing to localization of a membrane of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a receptor on the cell surface. When a substance or a substance capable of interacting with the receptor on the cell surface is a peptide or polypeptide as an active ingredient, the function of a receptor containing a nucleic acid encoding the functional substance as an active ingredient or the function of Therapeutic or prophylactic agents for diseases in which the transmission of information through them causes onset or progression of symptoms and in infectious diseases in which interaction with Z or receptor causes infection are also included. That is, the functional substance or a nucleic acid encoding the functional substance may be a disease in which the function of the receptor or the signal transmission via the receptor causes the onset or progression of the symptom, and the interaction with Z or the receptor may cause the infection. It can be used to treat or prevent the causative infection.

 [0081] Such a therapeutic or prophylactic agent can be used as an amount of the functional substance or a nucleic acid encoding the functional substance to be localized on the cell membrane surface of an individual at a disease site or at a site where a disease is likely to develop. A sufficient amount may be administered to the individual. Such a dose can be appropriately set according to, for example, the weight and age of the individual, the type and condition of the target disease or infectious disease, and the like. The same applies to the number of administrations and the form of administration.

[0082] Further, the functional substance or a nucleic acid encoding the functional substance may be a disease in which the function of the receptor or the signal transmission via the receptor causes the onset or progress of the symptom, and the interaction with the Z or the receptor. It can be used in the manufacture of a medicament for the treatment or prevention of infectious diseases that cause infection. [0083] Since the present invention is characterized in that a substance that interacts with a receptor is localized on the surface of a target cell, the effect is lost by diffusion as in a conventional control method using a soluble substance. It is useful for the continuous control of receptors that are not reported. In addition, by selecting a method having high specificity for a target cell as a method for introducing into a cell, it is possible to control the function of a cell-specific receptor without affecting the receptors of other cells. Become.

 Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited to these examples.

 Example 1

 Preparation of Retrovirus Vector Having gpl20 on the Surface

 A retrovirus vector having an HIV-derived envelope (env) on the virus surface and capable of introducing a nucleic acid encoding red-shift Green Fluorescent Protein (hereinafter referred to as rsGFP) into cells was prepared.

 [0086] First, the nucleic acid encoding rsGFP inserted into the plasmid pQBI25 (trade name, manufactured by Quantum Biotechnologies Inc.) was isolated. The obtained fragment was inserted into pDON-AI vector I (Takara Bio Inc.) to prepare a plasmid pDON-AI-rsGFP.

 [0087] The HIV envelope expression vector (pHIV env) was constructed as follows.

[0088] Trade name: An amphoter-pick pick envelope expression vector pE-ampho (trade name), which is a vector contained in Retro virus Packaging Kit Ampho (manufactured by Takara Bio Inc.), is digested with restriction enzymes EcoRI and Xbal. Then, the vector was removed from the pick envelope gene to prepare a vector monoskeleton fragment. On the other hand, pAbT4674 (ATCC No. 40829), which is an HIV envelope expression vector, was designated as type I, and a 5 'primer (SEQ ID NO: 2) and a 3' primer having a restriction enzyme Xbal recognition site (SEQ ID NO: 3) ) And PCR was performed to prepare an amplified fragment. The resulting amplified fragment was digested with restriction enzymes Hindlll and Xbal. The obtained product and an approximately 1.9 kb fragment obtained by digesting the pAbT4674 with the restriction enzymes EcoRI and Hindlll were ligated to the vector backbone fragment. The nucleotide sequence of the obtained vector was examined, and it was confirmed that the target DNA was inserted. The resulting vector was called pHIV env. [0089] 293T cells [Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 8392-8396 (1993;)] were added to 10% by volume of fetal serum ( ⁶ × 10 ⁶ Zml) to give a volume of 6 ml. The suspension was suspended in Dulbecco's modified Eagle's medium (DMEM) containing 100 μg / ml streptomycin and 100 units of Zml penicillin. 1 ml of the obtained cell suspension was added to each of five 10 cm gelatin-coated dishes containing 9 ml of the same medium. After 24 hours of culture, the culture supernatant is removed, a DMEM 7. 5 ml containing a 10 volume _^0/0 FBS and 25 mu Micromax black port kin, was added to the dish.

 [0090] 25 g of the pDON-AI-rsGFP and the trade name: Retrovirus Packaging Kit

 A mixture of 12.5 μg of pGP, a vector contained in Ampho (manufactured by Takara Bio Inc.), the above-mentioned pHIV env 12. and 2M CaCl1551, was mixed to obtain a mixture of 12501.

 2

 It was. To the obtained mixture, 1250 / zl of Tmnsfection buffer attached to the above kit was added to form a calcium phosphate precipitate, and then added to each of the dishes containing the above 293T cells.

[0091] After culturing the obtained cells in a 5 volume ⁰ / oCO incubator at 37 ° C for 7.5 hours, 10

 2

 The medium was replaced with 8 ml of DMEM containing the amount% FBS. After culturing for 24 hours from the transfection, the medium was replaced with 8 ml of DMEM containing 10% by volume of FBS. After further culturing for 24 hours, the culture supernatant was collected and filtered with a 0.45 m filter. The obtained filtrate was centrifuged at 4 ° C. and 2500 × g for 16 hours to obtain a virus precipitate. The virus precipitate was suspended in 2 ml of DMEM containing 10% by volume of FBS to obtain a virus solution (gpl 20-rsGFP virus).

[0092] The fibronectin fragment (trade name: RetroNectin, manufactured by Takara Bayo) was diluted with PBS (phosphate-buffered saline) to a concentration of 40 µg / ml. The resulting solution was cultivated in a 24-well plate at 400 1Z-well and left at 4 ° C. Thereafter, the solution was removed from the well, PBS containing 2% by weight of serum albumin (BSA) was added to the well in a 500 µl Z well, and the plate was allowed to stand at room temperature for 30 minutes for blocking. The inside Ueru and wash with PBS 500 mu 1, the above gp l 20- rsGFP virus, 3. 7 X 10 3 cells / Ueru, 1. 2 X 10 ³ cells / © E Le, 0.4 10 ³ After adding 7 or 0.14 x 10 ³ z-wells, 37. Incubated for 4 hours in a 5% by volume CO incubator. After that, The virus solution was removed from the sample to obtain a virus adhesion plate.

 [0093] MOLT-3, a human T cell line that expresses CD4 on the cell surface, was placed on a virus adhesion plate.

(JCRB9048), HeLaZCD4Q [. Virol., Vol. 62, pp. 3779-3788 (1988)] derived from human epithelial cell carcinoma-derived HeLa cells was calorificed to give 2 × 10 ⁴ cells / well. After culturing for 48 hours, the cells were collected and centrifuged at 300 X g for 5 minutes at room temperature. The cell pellet was washed three times with PBS containing 0.1% by weight of sodium azide and 0.5% by weight of BSA, and then subjected to analysis using a flow cytometer to measure the percentage of GFP-positive cells. Figure 1 shows the results.

 [0094] As shown in Fig. 1, gpl20-rsGFP virus infected MOLT-3 and HeLaZCD4, which are CD4 expression-positive cell lines, in a virus-dependent manner, and expressed rsGFP in the cells.

[0095] The 3. 7 X 10 ³ cells Z Ueru of gpl20- rsGFP virus the pre-chromatography bets adhered in a similar way, Do expressing CD4 expressing HeLa cells (HeLaZCD4) or CD4!, HeLa cells (ATCC CCL Fig. 2 shows the results when the virus infection was carried out after adding (2).

 [0096] As a result, as shown in Fig. 2, the CD4 positive cell line (HeLa / CD4) was infected. The CD4 negative cell line (HeLa) was not infected.

 Example 2

 [0097] Construction of plasmid for expressing HIV-derived gpl20 and mucin box-containing functional substance A rat neutral ceramidase mucin box expression vector (pCA-SM) was constructed as follows.

 The pE-ampho was digested with the restriction enzymes Kpnl and EcoRI to remove the Amphotopic Pickenbellus gene to prepare a vector backbone fragment.

[0098] On the other hand, the rat neutral ceramidase expression vector described in J. Biol. Chem., Vol. 276, pp. 26249-26259 (2001), pcDNAkCD, is a type I and has a 5 'side having a restriction enzyme Kpnl recognition site. PCR using a primer (SEQ ID NO: 4) and a 3 'primer having an EcoRI recognition site (SEQ ID NO: 5) encodes a signal sequence mutin box derived from rat neutral ceramidase with a flag-tag The DNA fragment to be amplified was amplified. The obtained DNA fragment was digested with restriction enzymes Kpnl and EcoRI. The resulting product was ligated to the vector backbone fragment. The nucleotide sequence of the obtained vector was examined, and it was confirmed that the target DNA was inserted. The resulting vector was designated as pCA-SM. pCA—SM There is a region encoding a signal sequence-mucin box derived from the rat neutral ceramidase gene with a flag-tag added downstream of the cytomegalovirus promoter, and downstream of it are EcoRI, EcoRV, There are recognition sites for NotI, Xhol, Xbal and Apal, respectively. According to the above-mentioned pCA-SM, the product of the target gene can be expressed as a fusion protein with the flag-tag signal sequence mucin box by inserting the target gene into the pCAM-SM in the same frame.

[0099] The pHIV env described in Example 1 was defined as type I, and a 5 'primer having a restriction enzyme EcoRI recognition site (SEQ ID NO: 6) and a 3' primer having an Xhol recognition site (SEQ ID NO: 7) PCR was performed using the DNA and DNA to obtain a DNA fragment encoding a portion containing HIV-derived gp120. The resulting product was subcloned into the EcoRI recognition site and the Xhol recognition site of the pCA-SM to obtain a plasmid (pCA-SMgp120) for expressing a mucin box gp120 fusion polypeptide.

 [0100] The plasmid (pCA SMgpl20) encodes a polypeptide having the amino acid sequence of SEQ ID NO: 8. In the amino acid sequence shown in SEQ ID NO: 8, the 2nd to 9th amino acids are ¾ag-tag, the 10th to 92nd amino acids are the rat neutral ceramidase-derived signal sequence mucin box region (the 51st to 86th amino acids are SEQ ID NO: : 1 mucin box).

 [0101] Further, as a plasmid expressing only gp120, a DNA fragment encoding a portion containing gp120 amplified in the construction of pCA SMgpl20 described above was used as a neomycin resistance gene expression plasmid pTriEx3-Neo [Novagen (Novagen)] and an Xhoi recognition site to construct a gp120 expression plasmid, pTriEx3-gpl20.

 Example 3

 [0102] Gene transfer of gp120 mucin box expression plasmid into cells

HeLa cells or HeLaZCD4 cells were transfected using 2 μg each of pCA-SMgpl20, pTriEx3-gpl20, pcDNAkCD and pTriEx3-NEO obtained in Example 2 and trade name: LipofectAMINE2000 (Invitrogen) 31. Yeong. HeLa a cell, HeLaZCD4 cells 10 volume _^0/0 FBS and, 100 mu g / ml streptomycin If, using DMEM containing 100 units Zml penicillin, 5 volumes of 37 ° C _^0/0 CO in

 2 Cultured in a cuvette.

 [0103] Twenty-four hours after the transfection, the cells were collected and centrifuged at 400 X g for 5 minutes at room temperature. The cell pellet obtained by centrifugation was suspended in the medium and used for the following experiments.

[0104] from the cell suspension by preparative 2 X 10 ⁵ cells cells min, and centrifuged 300 X g, 3 min. An anti-gpl20 antibody (manufactured by ICN Biomedicals, Inc.) or a FITC-labeled anti-CD4 antibody (manufactured by dithirene earth) solution 1001 was added to the cell pellet, and the mixture was incubated on ice for 30 minutes. Thereafter, the obtained product was washed twice with PBS 5001 containing 0.5% by weight of BSA and 0.1% by weight of sodium azide. With respect to the anti-gp120 antibody reaction sample, a FITC-labeled anti-mouse IgG antibody (manufactured by DAKO) 1001 solution was further added to the sample, the mixture was incubated on ice for 30 minutes, and then washed twice as described above. The sample after the antibody reaction was subjected to analysis using a flow cytometer, the number of FITC-positive cells was measured, and the gpl20-expressing cell rate and the CD4-expressing cell rate were measured. FIG. 3 shows the results of the anti-gp120 antibody, and FIG. 4 shows the results of the anti-CD4 antibody.

 [0105] As shown in Fig. 3, gpl20 was expressed on the cell surface of HeLa cells into which pCA-SMgpl20 had been introduced. No expression of gp120 was observed on the cell surface into which a plasmid other than pCA-SMgp120 was introduced.

 As shown in FIG. 4, in HeLaZCD4 cells into which pCA-SMgpl20 had been introduced, the amount of cell surface CD4 detected by the anti-CD4 antibody was reduced. That is, it became clear that the binding of the anti-CD4 antibody to CD4 was inhibited by the localization of gp120 on the cell surface by the expression of the mucin box gp120 fusion polypeptide.

 Example 4

 [0107] Virus infection of transfected cells

HeLaZCD4 cells were transfected using pCA-SMgp120, pcDNAkCD and pTriEx3-NEO obtained in Example 2 and trade name: LipofectAMINE2000 (Invitrogen), and cultured for 24 hours. The above-mentioned transfection was carried out on a plate to which 3.7 × 10 ³ gpl20-rsGFP virus had been adhered, prepared in the same manner as in Example 1. The cells after crushing were added to each well at 2 × 10 ⁴ cells / well, and cultured in a 5% by volume CO incubator at 37 ° C.

 2

 [0108] 48 hours after the virus infection, the cells were peeled and collected with 0.05% by weight of trypsin 0.53 mM EDTA and centrifuged at room temperature at 300 X g for 5 minutes. The obtained cell pellet was washed three times with PBS containing 0.1% by weight of sodium azide and 0.5% by weight of BSA. After that, the GFP-positive cell ratio was measured using a flow cytometer. Figure 5 shows the results of measuring the GFP-positive cell rate.

 [0109] As shown in Fig. 5, it was revealed that the virus infection rate was reduced in HeLaZCD4 cells into which the nucleic acid encoding the mucin box gpl20 (pCA SMgpl20) had been introduced. This indicated that the expression of the mucin box gpl20 fusion polypeptide suppressed the interaction between CD4 and gpl20-rsGFP virus.

 Industrial applicability

[0110] According to the present invention, a polypeptide that is not naturally localized on the cell membrane surface is localized on the cell membrane surface to cause an interaction between a receptor present on the cell surface and the polypeptide. Thus, the binding between the receptor and its agonist can be regulated or inhibited.

 Sequence listing free text

 [Oil 1] SEQ ID NO: 2 shows the sequence of a designed oligonucleotide PCR primer for amplifying an HIV envelope gene portion.

[0112] SEQ ID NO: 3 shows the sequence of a designed oligonucleotide PCR primer for amplifying a portion of HIV gp120 gene. Nucleotides 6-11 are the Xbal restriction enzyme recognition site.

 [0113] SEQ ID NO: 4 shows the sequence of a designed oligonucleotide PCR primer for amplifying a neutral ceramidase portion derived from Rattus norvegicus. Nucleotides 519 are the Kpnl restriction enzyme recognition sites.

[0114] SEQ ID NO: 5 shows the sequence of a designed oligonucleotide PCR primer for amplifying a neutral ceramidase portion derived from Rattus norvegicus. Nucleotides 519 are the EcoRI restriction enzyme recognition sites. [0115] SEQ ID NO: 6 is a designed oligonucleotide PCR primer sequence for amplifying a portion of HIV gp120 gene. Nucleotides 116 are EcoRI restriction enzyme sites.

 [0116] SEQ ID NO: 7 shows the sequence of a designed oligonucleotide PCR primer for amplifying a portion of HIV gp120 gene. Nucleotides 116 are the Xhol restriction enzyme recognition site.

 [0117] SEQ ID NO: 8 shows a sequence of an artificial protein containing a signal sequence, a mucin box of neutral ceramidase derived from Rattus norvegicus, and an HIV gp120 gene product.

Claims

The scope of the claims

 [1] Localizes a functional substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance that can interact with a cell surface receptor to the cell membrane surface A method for regulating the function of a receptor present on a cell surface, characterized in that:

 [2] The method according to claim 1, which inhibits an interaction between a receptor present on a cell surface and an extracellular substance.

 [3] The method according to claim 1, wherein the polypeptide localized on the cell membrane surface is a polypeptide containing a mucin box.

[4] The method according to claim 1, which is a peptide or polypeptide capable of interacting with a cell surface receptor.

 [5] The functional substance according to claim 4, wherein the functional substance is a fusion polypeptide of a region of the polypeptide localized on the cell membrane surface that contributes to the localization of the membrane and a polypeptide capable of interacting with a receptor on the cell surface. the method of.

 [6] Introduce into a cell a nucleic acid encoding a fusion polypeptide of a region contributing to membrane localization of the polypeptide localized on the cell membrane surface and a polypeptide capable of interacting with a receptor on the cell surface 6. The method of claim 5, wherein:

 [7] Substances capable of interacting with cell surface receptors Growth factors, cytokins, chemokines, peptide hormones, polypeptide hormones, cell adhesion factors, toxins, antibodies, microorganism-derived proteins, virus-derived proteins and 5. The method according to claim 4, which is a substance selected from the group consisting of these fragments.

 [8] The method according to claim 6, wherein the substance is a protein of a virus outer membrane capable of interacting with a cell surface receptor.

 [9] Localizes on the cell membrane surface a functional substance containing a region that contributes to the localization of the polypeptide localized on the cell membrane surface and a substance that can interact with the cell surface receptor A method for regulating signal transduction in an animal.

[10] A functional substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a receptor on the cell surface is transferred to an individual at the site of disease or disease. Localization on the cell membrane surface at the site of potential onset, A method for treating or preventing a disease in which the function of a puter or information transmission through it causes onset or progression of symptoms and an infectious disease in which an interaction with Z or a receptor causes infection.

 [11] A functional substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a receptor on the cell surface, or a nucleic acid encoding the functional substance, A therapeutic or prophylactic agent for a disease that is contained as an active ingredient and that causes the onset or progression of the function of the receptor or the transmission of information through it, and an infectious disease that causes infection by interaction with Z or the receptor. .

 [12] Cell membrane surface for the treatment or prevention of diseases in which the function of the receptor or signal transmission through it causes development or progression of symptoms, and infectious diseases in which interaction with Z or the receptor causes infection. Use of a functional substance containing a region contributing to localization of a polypeptide membrane localized in a membrane and a substance capable of interacting with a receptor on a cell surface, or a nucleic acid encoding the functional substance.

 [13] Manufacture of a drug for treating or preventing a disease in which the function of the receptor or signal transmission through it causes the onset or progression of symptoms and an infectious disease in which the interaction with Z or the receptor causes infection. Substance containing a region contributing to localization of a polypeptide localized on the cell membrane surface and a substance capable of interacting with a receptor on the cell surface, or a nucleic acid encoding the functional substance Use of.