JP2007129942A - Peptide binding to m (membrane) protein of sars coronavirus and inhibiting binding of m protein to n (nucleocapsid) protein and system for analyzing interaction of m protein with n protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance inhibiting binding of M protein of SARS (severe acute respiratory syndrome) coronavirus to N protein and inhibiting the formation of virus particles and to provide a method for screening the substance. <P>SOLUTION: The method for screening the substance inhibiting the binding of the M protein of the SARS coronavirus to the N protein is carried out as follows. A candidate substance inhibiting the binding of the N protein or the M protein or a partial peptide thereof which can be bound to a protein or a peptide changed into a solid phase to the M protein or the N protein is added to a peptide having a specific amino acid sequence and the M protein or the N protein or the partial peptide thereof changed into the solid phase on a substrate. Thereby, the substance inhibiting the binding of the N protein or the M protein or the partial peptide thereof that can be bound to the protein or the peptide changed into the solid phase is selected as the substance inhibiting the binding of the M protein to the N protein. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an N protein-M protein interaction analysis system derived from SARS coronavirus, a peptide that inhibits the interaction between M protein and N protein, the peptide, and the structure information of the peptide. The present invention relates to the treatment, diagnosis and prevention of the above-mentioned viral infections using the prepared compounds and antibodies recognizing the peptides.

  Severe acute respiratory syndrome (SARS) involving SARS coronavirus is a highly fatal disease, but the mechanism of its progression is unclear. Viral particle formation essential for the growth of SARS coronavirus (SARS-CoV) requires the interaction of the M protein and N protein of the virus (Non-patent Document 1). The M protein is a protein expressed on the surface of the virus particle and is suitable as a target for virus detection. It has been reported that antibodies that recognize partial peptides of M protein or N protein exist in SARS virus patients (Patent Document 1).

  However, a system for accurately analyzing the interaction between M protein and N protein in SARS coronavirus has not been constructed.

JP 2005-263773 A Huang Y, Yang ZY, Kong WP, Nabel GJ.Generation of synthetic severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production.J Virol. 2004 78 (22): 12557-65.

  The prior art has suggested that the interaction of M protein and N protein is important for SARS-CoV particle formation. However, from the conventional knowledge, the binding mode of the M protein and the N protein, for example, which part of each protein is involved in the binding has not been clarified. The present inventor considered that proliferation after infection with SARS-CoV can be suppressed by inhibiting the interaction between M protein and N protein. The present invention provides a method for analyzing the interaction between M protein and N protein, and a method for screening a virus particle formation inhibitor using the same method, a virus particle formation inhibitor peptide, an antibody that recognizes the peptide, or structure information of the peptide The purpose is to provide a compound prepared based on the above, and to provide a diagnosis, prevention and treatment method for SARS coronavirus infection using the peptide, an antibody recognizing the peptide or a compound prepared based on the structural information of the peptide And

  In order to elucidate the pathogenesis of SARS caused by SARS coronavirus and to establish a diagnosis, prevention, and treatment method, the present inventors have utilized an M protein partial peptide to which an antibody derived from a SARS coronavirus-infected patient binds. The present invention has been completed by establishing an M protein and N protein interaction analysis system.

  The present invention is a peptide comprising the amino acid sequence represented by SEQ ID NO: 12, or a peptide comprising the amino acid sequence represented by SEQ ID NO: 12, and not a full-length N protein. The peptides of the present invention also include modified derivatives to which other peptides are added in order to improve stability when administered in vivo, or to deliver efficiently to target cells, or for other reasons advantageous for treatment. included.

  Furthermore, the present invention includes a polynucleotide encoding a peptide comprising the amino acid sequence represented by SEQ ID NO: 12, an expression vector containing the polynucleotide, and the expression vector for providing a compound such as the above peptide. A host cell.

  A method for producing a peptide comprising the amino acid sequence represented by SEQ ID NO: 12 by recombinant DNA technology includes a step of culturing a host cell transformed with the above-mentioned vector containing a polynucleotide, and a peptide produced by the host cell. It is necessary to recover and purify the product. The present invention includes each of these processes and manufacturing methods.

  Furthermore, the present invention is a method for analyzing the interaction between N protein and M protein, and a method for screening an interaction inhibitor applying this method. As an example, there is a method for screening a substance that inhibits the binding between the M protein and N protein of SARS coronavirus, wherein the protein immobilized on the M protein or N protein immobilized on the substrate or a partial peptide thereof, or N protein or M protein capable of binding to peptide or partial peptide thereof, M protein and N protein binding inhibition candidate substance is added, and immobilized protein or peptide and immobilized protein or peptide can be bound to N A method for screening a substance that inhibits the binding of M protein and N protein of SARS coronavirus, wherein a substance that inhibits binding of protein or M protein or a partial peptide thereof is selected as a binding inhibitor of M protein and N protein. is there. Examples of the peptide immobilized on the substrate include a peptide including the amino acid sequence represented by SEQ ID NO: 12 and a peptide including the amino acid sequence represented by SEQ ID NO: 12, excluding the full-length N protein. Screening can be performed by ELISA or surface plasmon resonance. For example, there is a method including the following steps.

1) A plurality of peptides having 10 to 20 amino acids covering the entire length of M protein or N protein are immobilized on a substrate, and an antibody against N protein or M protein produced in a patient infected with SARS virus is recognized. Adding a full-length or partial peptide of N protein or M protein that can
2) a step of detecting an N protein or M protein bound to a solid-phased M protein or a partial peptide of the N protein, or a partial peptide thereof, using the SARS virus-infected patient-derived antibody used in the step 1);
3) As a result of the step 2), a solid phase N protein partial peptide or a partial peptide of the M protein to which the added M protein or N protein is most bound is used as a candidate peptide for an inhibitor of M protein and N protein. 4) M protein or N protein or a partial peptide thereof immobilized, and the N protein or M protein or their partial peptide to which the antibody used in the step 2) binds and the inhibitor identified in the step 3) A candidate peptide is added, and the candidate substance that inhibits the binding of M protein or its partial peptide and N protein or its partial peptide is selected as M protein using the antibody used in step 2). A step of forming an N protein binding inhibitor.

  Furthermore, the present invention is a SARS-CoV M protein and N protein binding inhibitor identified by the above method.

  Inhibiting substances such as a peptide containing the amino acid sequence represented by SEQ ID NO: 12 when screening for a binding inhibitor of M protein and N protein by the method for identifying a substance that inhibits binding of M protein and N protein as described above And can be used as a positive control substance.

  Furthermore, the present invention is a pharmaceutical composition for treating a patient infected with SARS-CoV, comprising as an active ingredient a substance that inhibits the binding of the above-mentioned M protein and N protein. By administering this pharmaceutical composition into a patient infected with SARS-CoV, binding of M protein and N protein is inhibited. As a result, inhibition of SARS-CoV particle formation can be expected to suppress the growth of the virus in the patient.

Furthermore, the present invention is an anti-SARS-CoV DNA vaccine comprising a vector comprising the polynucleotide of the present invention as a composition for treatment applied to SARS-CoV infected patients. Details of the vaccine are described in Example 3.
Furthermore, the present invention is a pharmaceutical composition for gene therapy comprising the above expression vector.

  The M protein and N protein interaction analysis system of the present invention and the peptide that inhibits the binding of the M protein and the N protein can be used for research, diagnosis and treatment of diseases related to SARS-CoV. It can be used for the development of diagnostic kits, therapeutics and therapeutics.

  In particular, as described in Example 4 below, as an effect on treatment, by using the peptide of the present invention, binding of M protein and N protein can be inhibited, and virus particle formation of SARS-CoV can be inhibited. Results It can be expected to inhibit SARS-CoV proliferation. In addition, as described in Example 3, a DNA encoding the peptide of the present invention was inserted into a vector and administered to a mammal infected with SARS-CoV to form an antibody against N protein in vivo, and SARS -Expected to have therapeutic effects on diseases involving CoV.

  The substance that inhibits the binding between the M protein and the N protein of the present invention is a substance that binds to the M protein or the N protein, or a site that binds to the M protein, and competitively inhibits the binding between the M protein and the N protein. It is. The type of the substance is not limited, but for example, it is a peptide that inhibits the binding of M protein and N protein, binds to the binding site of M protein or N protein with N protein or M protein, respectively, and competes with M protein. It is a peptide that inhibits the binding of N protein. Examples of such a peptide include a partial peptide that binds to the binding site of the M protein or N protein with the N protein or M protein, respectively. An example is a peptide comprising the amino acid sequence represented by SEQ ID NO: 12. Moreover, the peptide of this invention is a peptide containing the amino acid sequence represented by sequence number 12, and is a peptide except the peptide which consists of a full length sequence of N protein. As such a peptide, for example, a continuous amino acid sequence of the amino acid sequence of the N protein represented by SEQ ID NO: 1 and the number of amino acid residues including the portion represented by SEQ ID NO: 12 from 15 to 421, 15 400, 15 to 350, 15 to 300, 15 to 250, 15 to 200, 15 to 150, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, Peptides having an amino acid sequence of 15 to 30, 15 to 25, or 15 to 20 are included.

  Further, in the peptide comprising the amino acid sequence represented by SEQ ID NO: 12 of the present invention and comprising a partial amino acid sequence of the N protein amino acid sequence represented by SEQ ID NO: 1 other than the amino acid sequence represented by SEQ ID NO: 12 In this amino acid sequence, one or several amino acids may be deleted, substituted or added. A deletion, substitution or addition of one or several amino acids refers to a deletion, substitution or addition of 1 to 10, preferably 1 to 5, more preferably 1 or 2 amino acids.

  The above-mentioned inhibitory peptide for binding of the M protein and N protein of the SARS coronavirus of the present invention is produced not only by the peptide synthesis method described above but also by using host cells transformed using a known recombinant DNA technique. be able to. For this purpose, a recombinant nucleic acid molecule of DNA or RNA encoding the peptide is prepared. Methods for producing recombinant DNA and / or RNA molecules are known in the art. For example, a base sequence encoding the peptide of the present invention can be excised from DNA using an appropriate restriction enzyme, and a related sequence inserted with a restriction site useful for subsequent cloning can be prepared by polymerase chain reaction (PCR). it can. The DNA / RNA molecule encoding the peptide of the present invention can be synthesized using a chemical synthesis technique such as the phosphoramidite method.

  The present invention also includes a vector containing DNA encoding a peptide that inhibits the binding of M protein and N protein, and a host cell containing the vector. The vector includes a DNA molecule encoding a peptide of the invention operably linked to suitable expression control sequences. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, initiation signals, termination signals, cap signals, polyadenylation signals, and other signals involved in the regulation of transcription or translation. The control sequence can be linked to the DNA encoding the peptide of the present invention and incorporated into a vector by a known method.

  An appropriate host can be transformed with a vector containing a DNA molecule encoding the peptide of the present invention. Transformation can be performed using known methods.

  As the host cell, a number of known host cells that are commercially available can be used. The host cell may be selected depending on a plurality of factors. These factors include, for example, compatibility with selected expression vectors, toxicity of peptides encoded by DNA molecules to host cells, transformation efficiency, ease of peptide recovery, expression characteristics, biosafety and cost. included. DNA molecules can be efficiently expressed by appropriately changing these factors depending on the host.

  Microbial hosts used in the present invention include bacteria (eg, E. coli), yeasts (eg, Saccharomyces sp. And Pichia pastoris), fungi, insects, plants, mammalian (including human) cells and other hosts known in the art. Is included. The transformed cells may be cultured under known fermentation conditions so that the desired peptide is expressed. Thereafter, the peptide can be purified by known methods from fermentation cultures or host cells in which the peptide is expressed.

  The present invention further includes a method of identifying a substance that inhibits the binding of SARS coronavirus M protein and N protein. The method can be performed using a peptide library derived from N protein or M protein. Here, the peptide library refers to a collection of partial peptides covering the full-length amino acid sequence of the M protein or N protein, and is designed to have 6 to 20 amino acids designed to overlap several amino acids at both ends of the peptide, preferably It consists of a partial peptide of 10 to 20 amino acids. For example, for N protein, a peptide library consisting of 42 types of peptides of 15 amino acids can be used. Each peptide in the peptide library can be used for ELISA immobilization.

  The antibody used in the method for identifying a substance that inhibits the binding of the M protein and N protein of the SARS coronavirus of the present invention is an M protein or a partial peptide of the N protein, which is produced in a patient infected with SARS coronavirus Is an antibody that recognizes a partial peptide recognized by The partial peptide recognized by the antibody is an M protein or N protein, or a complex of the partial peptide and the antibody is M protein or a partial peptide thereof-N protein or a partial peptide-antibody thereof, or N protein or a partial peptide thereof-M. It is not limited as long as it is formed in the form of a protein or a partial peptide-antibody thereof. The reactivity of a blood sample collected from a patient infected with SARS-CoV and several types of M protein partial peptides described in JP-A-2005-263773 is examined, and the partial peptide recognized by the antibody present in the patient is immunogen. As described above, an antibody used in the method of the present invention can be prepared. The antibody may be a polyclonal antibody or a monoclonal antibody, and can be prepared by a known method. Examples of such an antibody include an antibody that recognizes the C-terminal partial peptide (M63) in the M protein fragment peptide of SARS coronavirus. The amino acid sequence of the M protein of SARS coronavirus is shown in SEQ ID NO: 44.

  The above N protein-derived peptides and the peptides of the present invention can be synthesized according to methods commonly used in peptide chemistry. Examples of known methods include “Peptide Synthesis”, Interscience, New York, 1996; “The protein”, vol. 2, Academic Press Inc., New York, 1976; “Peptide Synthesis” (Maruzen Co., 1975), etc. It is described in the literature.

  Identification of a peptide that inhibits the binding of the M protein and N protein of the SARS coronavirus of the present invention is performed, for example, as follows. Each peptide of the peptide library of M protein or N protein is immobilized on an ELISA microplate, and if the peptide library is derived from M protein, the N protein or its partial peptide is immobilized on the immobilized peptide. When is derived from N protein, M protein or a partial peptide thereof is reacted. At this time, a partial peptide of N protein or a partial peptide of M protein to be bound to each peptide of the peptide library is a partial peptide that produces a specific antibody in human body when SARS coronavirus is infected to human, It is a partial peptide containing an epitope. Such a peptide can be obtained by selecting from the above peptide library a peptide that reacts with an antibody against SARS coronavirus present in the blood of a human infected with SARS coronavirus. Subsequently, an antibody that recognizes a partial peptide of the above-mentioned M protein or N protein that is recognized by an antibody produced in a patient infected with SARS coronavirus is added. At this time, the antibody is labeled with an enzyme such as alkaline phosphatase or horseradish pepoxidase. Next, a substrate is added, an enzyme reaction is caused, and the peptide solid-phased in the well where color development is recognized is defined as a binding inhibitory peptide of M protein and N protein.

  Furthermore, the binding inhibitory peptide of other M protein and N protein can be identified using this binding inhibitory peptide. The binding inhibitory peptide is immobilized on an ELISA microplate, and the M protein or N protein or a partial peptide thereof and a binding inhibitor candidate substance are added. Further, an antibody that recognizes the M protein or N protein of SARS coronavirus and labeled with an enzyme is added, and the color is developed by enzymatic action as described above. Substances that inhibit the binding of the binding inhibitory peptide to the M protein or N protein or their partial peptides can be identified as a binding inhibitor of M protein and N protein. The present invention is also a method for screening for binding inhibitors of M protein and N protein. Examples of the binding inhibitor of M protein and N protein other than peptides include low molecular weight compounds.

In the above example, ELISA using an enzyme-labeled antibody is described in detail. However, binding of immobilized M protein or N protein or their partial peptide to N protein or M protein or their partial peptide Any method other than ELISA may be used as long as it can measure the above. In this case, the M protein or N protein or a partial peptide thereof may be used by immobilizing it on various substrates. In addition, for detection, an antibody labeled with a fluorescent substance or a radioisotope can be used, and as described below, the binding of M protein and N protein can be detected by a surface plasmon resonance method or the like without using an antibody. You can also.
The ELISA method and the solid phase method itself are described in detail in, for example, Clinical Examination, Vol. 47, No. 13, page1611-1618.

  The present invention includes a method and system for analyzing the binding of SARS coronavirus M protein and N protein by surface plasmon resonance (SPR).

  SPR utilizes the fact that an evanescent wave is generated by a polarized light beam irradiating a metal and oozes on the surface, excites surface plasmons, which are surface waves, consumes light energy, and reduces reflected light intensity. The resonance angle at which the reflected light intensity is significantly reduced varies depending on the thickness of the layer formed on the metal surface. Therefore, the substance to be measured can be fixed on the surface of the metal thin film, and the interaction with the substance in the sample can be detected by measuring the change in the resonance angle or the change in the reflected light intensity at a certain angle. Therefore, SPR does not require labeling with fluorescent materials, radioactive materials, and the like. SPR can be performed using a commercially available SPR detector.

  When performing bond analysis using SPR, it is necessary to use a substrate on which a metal thin film is formed. As the metal, gold, silver, copper, aluminum, platinum, or the like can be used, and these may be used alone or in combination to form a thin film on the substrate. The method for forming the metal thin film is not limited, and any known method such as vapor deposition, sputtering, or ion coating may be used. Among these, the method by vapor deposition is preferable. The thickness of the metal thin film is 10 to 3000 mm, preferably 100 to 600 mm. A known substrate is preferably transparent, and a substrate made of a resin such as glass, polyethylene terephthalate, polycarbonate, or acrylic may be used. The thickness of the substrate used for SPR is about 0.1 to 20 mm, preferably about 1 to 2 mm. In SPR, in order to obtain a reflection image from a metal thin film, it is desirable that the SPR resonance angle is small. A SARS coronavirus M protein or N protein or a partial peptide thereof is immobilized on a substrate on which a metal thin film is formed. In order to immobilize the peptide on the substrate, a functional group such as an amino group, a carboxyl group, a mercapto group, or an aldehyde group is introduced onto the substrate, and the functional group and the peptide are bonded to each other. Introduction of a functional group to the substrate and bonding of the peptide and the functional group can be performed by a known method. Furthermore, you may use the glass-made board | substrate with which the metal thin film for commercially available SPR was formed.

  By the above SPR, the binding between the immobilized M protein or N protein or a fragment thereof and the N protein or M protein or a fragment thereof can be analyzed. At this time, it is possible to measure whether or not a bond is formed by adding a binding inhibitor candidate substance of M protein and N protein and determine whether or not the binding inhibitor candidate substance has a function as a binding inhibitor. it can. That is, a binding inhibitor of M protein and N protein can be screened by the above SPR.

  Furthermore, the present invention provides a pharmaceutical composition comprising the SARS coronavirus M protein or N protein partial peptide of the present invention as an active ingredient, and a medicament comprising a substance that inhibits the binding of the SARS coronavirus M protein and N protein as an active ingredient. Includes the composition. At this time, it is necessary to cause the partial peptide of the present invention to act inside the cell.

  As a means of causing a peptide to act in cells, a method has been reported in which a protein transduction domain (hereinafter referred to as PTD) in a TAT protein of human immunodeficiency virus is bound to a peptide or protein desired to reach the cell (Cancer Research, 61: 474-477, (2001)). In addition to PTD in the TAT protein, various peptides that can deliver a desired peptide into cells are known, and these peptides can be used in the present invention. By binding PTD to the inhibitory peptide of the present invention and administering it to a patient infected with SARS-CoV, binding of intracellular M protein and N protein can be inhibited, and as a result, SARS-CoV proliferation can be suppressed.

  The inhibitory peptide or pharmaceutical composition of the present invention can be applied to patients infected with SARS-CoV. The diagnosis of SARS-CoV-infected patients is described in, for example, the literature influenza (Vol. 4, No. 4, p321-327). For such patients, the peptide of the present invention or a pharmaceutical composition containing the peptide can be used for therapeutic purposes.

  The pharmaceutical composition of the present invention comprises an effective amount of the peptide of the present invention and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Generally, the pharmaceutical composition comprises a buffer, a diluent having a specific pH and ionic strength (eg, Tris-HCl, acetate, phosphate), a detergent (eg, Tween 80, Polysorbate 80). And solubilizers)), antioxidants (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, timmersol, benzyl alcohol) or bulking agents (eg, lactose, mannitol). Furthermore, these substances may be incorporated into a granular preparation of polymer compounds (for example, polylactic acid, polyglycolic acid, etc.) or liposomes. In this case, hyaluronic acid can also be used, which may have the effect of extending the duration of the circulation. In addition, the pharmaceutical composition of the present invention may comprise other pharmaceutically acceptable pharmaceutical vehicles, liquids that function as excipients or vehicles, or semi-solid or solid diluents. These include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propyl-hydroxybenzoate, starch, sucrose, dextrose, acacia gum, calcium phosphate, mineral oil, cocoa butter and cocoa oil. Not. The substances contained in the pharmaceutical composition of the present invention can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the peptide of the present invention. See, for example, Remington's Pharmaceutical Sciences, 18th Edition, p.1435-1712, published by Mack Publishing Co. (1990), Easton, PA, which is incorporated herein by reference. The pharmaceutical composition of the present invention may be manufactured in liquid form, may be a dry powder (eg, lyophilized form), or may be an implantable sustained release formulation such as a subcutaneous formulation. .

  The pharmaceutical composition of the present invention is for example intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intraocular, intraocular, retrobulbar, intrapulmonary (eg, aerosolized drug) or (for extended release) By subcutaneous injection (including cumulative administration); via the sublingual, anal, vagina; or injection, including surgical implantation such as implantation in the subsplenial, subbrain or cornea, orally, nasally, transdermally or It can be administered in other dosage forms.

  In the present invention, pulmonary delivery of the peptide or pharmaceutical composition of the present invention is also contemplated. The polypeptide or a compound containing it is delivered to the lungs of a mammal while inhaling and is transported across the lung epithelial lining into the bloodstream. Pulmonary delivery is described in Adjei et al., Pharmaceutical Research, 7: 565-569 (1990); Adjei et al., International Journal of Pharmaceutics, 63: 135-144 (1990) (leuprolide acetate); Braquet et al., Journal of Cardiovascular Pharmacology, 13 ( Addendum 5): s.143-146 (1989) (Enserine-1); Hubbard et al., Anals of Internal Medicine, 3: 206-212 (1989) (1-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (1-proteinase); Oswein et al., “Aerosolization of Proteins”, Proceedings of the Symposium on Respiratory Drug Delivery held in Keystone, Colorado in March 1990 (Proceedings of Symposium of Respiratory Drug Delivery) II (recombinant human growth hormone); Debs et al., The Journal of Immunology, 140: 3482-3488 (1988) (interferon and tumor necrosis factor) and Platz et al. US Pat. No. 5,284,656. Based on the description (granulocyte colony stimulating factor) etc. Ukoto can.

  Administration by pulmonary delivery can also be performed using various mechanical devices designed for pulmonary delivery of therapeutic agents. Such devices include, but are not limited to, nebulizers, metered dose inhalers and powder inhalers well known to those skilled in the art.

  All such devices must use a formulation suitable for dispensing the pharmaceutical composition of the present invention. Typically, each formulation is specific to the type of device used, and a suitable propellant may be used in addition to the therapeutically useful diluent, adjuvant and / or carrier.

  Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose and sorbitol. Other ingredients used in the formulation may include DPEC, DOPE, DSPC and DOPC. It may also contain natural or synthetic surfactants. Furthermore, it may contain dextran such as polyethylene glycol and cyclodextran. In addition, bile salts and other related enhancers, cellulose and cellulose derivatives or amino acids as used in buffer formulations may be included.

  Liposomes, microcapsules or microspheres, encapsulated complexes or other types of carriers can also be used to administer the pharmaceutical composition of the invention.

  Dosage regimens for how to prevent or treat SARS coronavirus infections include various factors that alter the action of the drug, such as patient age, condition, weight, sex and diet, severity of infection, time of administration and other clinical factors To be determined by the attending physician.

  The pharmaceutical composition of the present invention can be administered by continuous infusion first to maintain therapeutic circulating levels of the drug after bolus injection. As another example, the pharmaceutical composition of the present invention can be administered as a single dose. One skilled in the art can readily optimize effective doses and dosing regimens determined by good medical practice and the clinical status of each patient. The administration frequency may be determined depending on the pharmacokinetic parameters of the drug and the administration route. The optimal pharmaceutical formulation will also be determined by one skilled in the art depending on the route of administration and the desired dose. See, for example, Remington's Pharmaceutical Sciences, 18th Edition, p.1435-1712, Mack Publishing Co. (1990), located in Easton, PA, incorporated herein by reference. The formulation can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the drug being administered. Depending on the route of administration, an appropriate dose can be calculated according to body weight, body surface area or organ size. The calculations necessary to determine the appropriate dose for treatment containing each of the above-described formulations are not limited to undue experimentation, particularly the dose information and assays described herein and the human clinical trials described above. One of ordinary skill in the art can routinely review further in light of the pharmacokinetic data found in the study. Appropriate doses can be determined using established assays for determining blood level doses and appropriate dose-response data. The final dosing regimen includes various factors that alter the action of the drug, such as specific activity of the drug, severity of damage and patient responsiveness, patient age, condition, weight, sex and diet, severity of infection, administration Determined by the attending physician taking into account the timing and other clinical factors.

  For example, an effective dose is generally desirably administered in the range of 1 ng to 1 mg / 1 kg body weight. In the treatment of SARS coronavirus infection, it may be administered once or multiple times over a period of time.

  Furthermore, a polynucleotide comprising the amino acid sequence represented by SEQ ID NO: 12 of the present invention or a peptide comprising the amino acid sequence represented by SEQ ID NO: 12 except for the full-length N protein is used for gene therapy. can do. For example, by expressing the peptide of the present invention in a living body, SARS coronavirus particle formation can be inhibited and SARS coronavirus infection can be treated. The gene of interest in gene therapy can be introduced into a subject by a known method. As a method for introducing a gene into a subject, there are a method using a viral vector and a method using a non-viral vector, and various methods are known (separate volume experimental medicine, basic technology of gene therapy, Yodosha, 1996; separate volume). Experimental Medicine, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997; edited by Japanese Society of Gene Therapy, Gene Therapy Research Handbook, NTS, 1999).

  Representative examples of viral vectors for gene transfer include methods using viral vectors such as adenoviruses, adeno-associated viruses, and retroviruses. Introduce the target gene into DNA virus or RNA virus such as detoxified retrovirus, herpes virus, vaccinia virus, pox virus, poliovirus, shinbis virus, sendai virus, SV40, immunodeficiency virus (HIV), It is possible to introduce a gene into a cell by infecting the cell with a recombinant virus. When the gene according to the present invention is used for gene therapy using a virus, an adenovirus vector is preferably used. Adenoviruses for gene therapy include first generation adenovirus vectors lacking the E1 / E3 region (Miyake, S., et al., Proc. Natl. Acad. Sci. USA., 93, 1320, 1996) from the second generation adenovirus vector in which E2 or E4 region is deleted in addition to E1 / E3 region (Lieber, A., et al., J. Virol., 70,8944, 1996; Mizuguchi, H. & Kay, MA, Hum. Gene Ther., 10, 2013, 1999), a third generation adenoviral vector (Steinwaerder, DS, et al., J) with almost complete deletion of the adenoviral genome (GUTLESS). Virol., 73, 9303, 1999). Any adenoviral vector can be used for gene therapy using the polynucleotide of the present invention. In addition, an adeno-AAV hybrid vector (Recchia, A., et al., Proc. Natl. Acad. Sci. USA., 96, 2615, 1999) or a transposon gene that has been incorporated into the AAV chromosome is used. Thus, it is possible to use an adenovirus vector having the ability to be integrated into a chromosome. It is also possible to deliver an adenoviral vector in a tissue-specific manner by inserting a tissue-specific peptide sequence into the H1 loop of adenoviral fiber (Mizuguchi, H. & Hayakawa, T., Nippon Rinsho, 7 , 1544, 2000).

  Moreover, it is also possible to introduce a target gene into cells or tissues using a recombinant expression vector in which a gene expression vector such as a plasmid vector is incorporated without using the virus. For example, the gene can be introduced into cells by lipofection method, phosphate-calcium coprecipitation method, DEAE-dextran method, direct DNA injection method using a micro glass tube. In addition, gene transfer method using internal liposome, gene transfer method using electrostatic type liposome, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), HVJ-E ( Envelope) vector method, receptor-mediated gene introduction method, method of transferring DNA molecule together with carrier (metal particle) with particle gun, naked-DNA direct introduction method, introduction method with various polymers, etc. It is possible to incorporate a recombinant expression vector into a cell. As the expression vector used in this case, any expression vector can be used as long as it can express the target gene in vivo. For example, pCAGGS (Gene 108, 193-200 (1991)), pBK -Expression vectors such as CMV, pcDNA3, 1, pZeoSV (Invitrogen, Stratagene), and pVAX1.

Construction of M protein and N protein binding detection system by ELISA, and screening of M protein and N protein binding inhibitor using both protein binding inhibitory peptides of the present invention as a positive control N protein-derived peptide (10 μg / mL, 50 μL, Diluted buffer; 50 mM NaCO ₃ pH 9.0) was immobilized on a 96-well microplate, blocked, and then recombinant M protein (1 μg / mL, 50 μL, diluted buffer; PBST) was added and bound. The 42 types of N protein-derived peptides used are shown in Table 1 below. Bound M protein was detected by anti-M protein antibody. When a certain amount of the peptide of the present invention was added to the M protein solution in advance and added to the N protein solid phase plate after incubation for 30 minutes, the N concentration of the peptide of the present invention was 0.01 μg, and the N concentration was 50%. Inhibition of protein binding to M protein was determined (FIG. 1).

Construction of binding analysis system between M protein and peptide or N protein of the present invention using surface plasmon resonance method (1) Binding analysis of N protein body or N protein-derived peptide to M protein by BIACORE
Recombinant M protein (100 μg / mL, diluted buffer; 10 mM Na-Acetate pH 5.0) was covalently bound to a sensor chip of BIACORE 3000 BIACORE3000 bioaffinity sensor (using an amine coupling kit). The N protein (dilution buffer; HBS-P) at the concentration shown in FIG. 2 was loaded on the same chip, and the dissociation constant of the binding of the N protein to the M protein was measured. The dissociation constant was calculated to be 2.5 nM. As for the buffer used in BIACORE3000, A was HBS-EP and B was HBS-P (manufactured by BIAcore).

(2) A peptide of the present invention comprising a control peptide or the amino acid sequence represented by SEQ ID NO: 12 (10 μg / mL, 70 μL, diluted) on the same sensor chip as that used in (1) to which a recombinant M protein is covalently bound buffer; HBS-P) was loaded and the binding was analyzed. As shown in FIG. 3, when the peptide was added to the M protein-immobilized sensor chip using the BIACORE3000 system (the uppermost line in FIG. 3), clear binding with the M protein was observed. The peptide of the present invention was added to the M protein-immobilized sensor chip at different concentrations using the BIACORE3000 system, and the dissociation constant between the M protein and the peptide of the present invention was measured and found to be 1.41 μM (FIG. 4).

Construction of Plasmid DNA Vaccine (pcDNA3.2-Npep) A DNA encoding the peptide of the present invention consisting of the amino acid sequence represented by SEQ ID NO: 12 was amplified by PCR, and the plasmid pCDNA3.2 / V5-DEST for mammalian cell expression ( Invitogen) was inserted directly downstream of the cytomegal virus enhancer / promoter region using the Gateway system (Invitrogen).

Mouse immunization The pcDNA3.2-Npep was introduced into Balb / C mice using a gene gun (Bio Rad Laboratories). 1 mg of gold particles was coated with 2 μg of the plasmid, and 0.5 mg of gold particles were shot twice into the mouse abdomen. The same operation was performed 4 times per week.

Evaluation of Mouse Immune Status Mice immunized with plasmid DNA vaccine were administered 50 μg of recombinant N protein from the tail vein. Serum was prepared from blood collected from the tail vein of the mice 5 days later.
Recombinant N protein was immobilized on a 96-well ELISA plate in 50 mM sodium carbonate buffer (pH 9.0) at 50 ng / well. After blocking with Superblock solution (Bio-rad) for 30 minutes, add 50 μl of mouse serum diluted 100, 1000 and 10000 times and control serum (collected from non-immunized mice), and incubate at room temperature for 1 hour. The amount of mouse immunoglobulin bound to the N protein was measured with an HRP-labeled anti-mouse IgG antibody. In the immunized mouse serum, a significant increase in antibody titer was observed even at 1000-fold dilution, but in the non-immunized mouse, no increase in antibody titer was observed.

Inhibition of SARS-CoV virus pseudoparticle formation by the peptide of the present invention By using the polypeptide of the present invention, the binding of N protein and M protein is inhibited. As a result, SARS-CoV virus particle formation inhibitory effect can be obtained. The following examples illustrate the invention but are not intended to limit the invention.

3 μg each of pcDNA3.2 / V5-DEST (Invitrogen), a plasmid for mammalian cell expression incorporating DNA encoding M protein, and pcDNA3.2 / V5-DEST (Invitrogen) incorporating DNA encoding N protein Each is introduced into 3 × 10 ⁶ 293 cells cultured in DMEM medium containing 10% FCS in a 10 cm Petri dish by a lipofection method using Fugene6 Transfection Reagent (Roche).

One day later, 50 μg of the peptide of the present invention is introduced into the aforementioned cells by a lipofection method using BioPORTER Reagent (Gene Therapy systems). As a control, lipofection without the peptide of the present invention is performed.
On the third day of culture, the medium is removed, 2.5 ml of PBS is added to the Petri dish, and the cells are destroyed by freezing and thawing three times.

After removing the cell debris from the resulting cell disruption solution by centrifugation at 1000 rpm for 5 minutes, 2.1 ml of this was mixed with 2.1 ml of OptiPrep (AXIS-SHIELD) and sealed in a tube for Beckman VTi65.1 ultracentrifuge rotor. Centrifuge at 350,000 xg for 3.5 hours.
After centrifugation, the tube is opened, and the content solution is fractionated 0.4 ml from the upper layer (fractions 1 to 11 respectively), 20 μl of each fraction is taken, and general SDS-PAGE and Western blotting are performed.

  Each protein is detected by a general method using an anti-N protein antibody or an anti-M protein antibody. When the peptide is not introduced, a small amount of N protein and M protein are detected in the fraction 1, and a large amount is detected mainly in the fractions 5-6. Detected.

  The M protein and N protein binding measurement system and peptide sequence of the present invention can be used for treatment of diseases involving SARS-CoV, development of therapeutic methods, and development of therapeutic agents.

It is a figure which shows the construction of the binding analysis system of M protein and N protein in ELISA, and specific inhibition of the binding of both proteins by the peptide of the present invention. Measurement of the dissociation constant of the interaction between M protein and N protein: It is a figure showing the result of measuring the dissociation constant of the binding of both proteins by adding N protein to the M protein-immobilized sensor chip at different concentrations in the BIACORE3000 system. . It is a figure which shows the specific coupling | bonding of this invention peptide to M protein. It is a figure which shows the result of the measurement of the dissociation constant of interaction of M protein and invention peptide.

Claims (16)

  A peptide comprising the amino acid sequence represented by SEQ ID NO: 12.   A peptide comprising the amino acid sequence represented by SEQ ID NO: 12, excluding the full-length N protein.   A polynucleotide encoding the peptide according to claim 2.   An expression vector comprising the polynucleotide according to claim 3.   A host cell comprising the expression vector according to claim 4.   A method for producing the peptide according to claim 1 or 2, comprising culturing a host cell transformed with the vector containing the polynucleotide according to claim 3, and recovering the polypeptide produced by the host cell. And the process of refine | purifying.   A method for screening a substance that inhibits the binding of M protein and N protein of SARS coronavirus, comprising M protein or N protein immobilized on a substrate or a partial peptide thereof; N protein or M protein capable of binding, or partial peptide thereof, M protein, and N protein binding inhibition candidate substance are added, and the immobilized protein or peptide and the immobilized N protein or peptide A method for screening a substance that inhibits the binding of M protein and N protein of SARS coronavirus, comprising selecting a substance that inhibits binding of M protein or a partial peptide thereof as a binding inhibitor of M protein and N protein .   The peptide of claim 1 or 2 is solid-phased on a substrate, and M protein or a partial peptide thereof, M protein and N protein binding inhibition candidate substance are added, and the SARS coronavirus of claim 7 is added. A method for screening a substance that inhibits the binding of M protein and N protein.   The method for screening for a substance that inhibits the binding of SARS coronavirus M protein and N protein according to claim 7 or 8, which is carried out by ELISA using an antibody that recognizes the M protein or N protein of SARS coronavirus.   A substance that inhibits the binding of M protein and N protein of SARS coronavirus according to claim 7 or 8, which is carried out by surface plasmon resonance using a substrate on which M protein or N protein or a partial peptide thereof is immobilized. How to screen. A method for screening a substance that inhibits the binding of M protein and N protein of SARS coronavirus, comprising the following steps:
1) A plurality of peptides having 10 to 20 amino acids covering the entire length of M protein or N protein are immobilized on a substrate, and an antibody against N protein or M protein produced in a patient infected with SARS virus is recognized. Adding a full-length or partial peptide of N protein or M protein that can
2) a step of detecting an N protein or M protein bound to a solid-phased M protein or a partial peptide of the N protein, or a partial peptide thereof, using the SARS virus-infected patient-derived antibody used in the step 1);
3) As a result of the step 2), the solid phase N protein partial peptide or M protein partial peptide to which the added M protein or N protein is most bound is used as a candidate peptide for an inhibitor of M protein and N protein. ,
4) M protein or N protein or a partial peptide thereof is solid-phased, N protein or M protein to which the antibody used in the step 2) binds or a partial peptide thereof, and an inhibitor candidate peptide identified in the step 3) And the candidate substance that inhibits the binding of M protein or its partial peptide and N protein or its partial peptide among the above-mentioned inhibitor candidate peptides using the antibody used in the step 2), M protein and N protein A step of forming a binding inhibitor.   The screening method of Claim 11 performed by ELISA.   A binding inhibitor of M protein and N protein of SARS coronavirus, selected using the method according to any one of claims 7 to 12.   A pharmaceutical composition for preventing or treating SARS virus infection, comprising the peptide according to claim 1 or 2 as an active ingredient.   14. A pharmaceutical composition for preventing or treating SARS virus infection, comprising as an active ingredient the binding inhibitor of SARS coronavirus M protein and N protein according to claim 13.   A pharmaceutical composition for gene therapy comprising the expression vector according to claim 4.

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