Protein, hemolytic streptococcus vaccine, DNA, vector, transformant, method for producing protein, baculovirus, agrobacterium, latex particle, kit, and method for measuring anti-streptolysin O antibody

Abstract

The present invention discloses any protein selected from among: (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 3; (b) a protein comprising an amino acid sequence represented by SEQ ID NO: 2; (c) a protein comprising an amino acid sequence represented by SEQ ID NO: 4; (d) a protein comprising an amino acid sequence represented by SEQ ID NO: 2, wherein some of the amino acid residues at positions 1-48 are deleted; (e) a protein comprising an amino acid sequence represented by SEQ ID NO: 4, wherein some of the amino acid residues at positions 380-490 are deleted; (f) a protein that comprises an amino acid sequence having at least 90% identity to the amino acid sequence of any one of the proteins (a)-(e) and that binds to an anti-streptolysin O antibody; and (g) any one of the proteins (a)-(f) wherein a tag is added to at least one of the C-terminal and the N-terminal thereof.

Description

[Technical field]

[0001] The present invention relates to a protein, a hemolytic streptococcal vaccine, DNA, a vector, a transformant, a method for producing a protein, a baculovirus, an Agrobacterium, a latex particle, a kit, and a method for measuring anti-streptolysin O antibody. [Background technology]

[0002] Streptolysin O (SLO) is a protein with hemolytic activity produced by Streptococcus pyogenes. When humans are infected with Streptococcus pyogenes, anti-SLO antibodies (ASO) in the blood increase. Using this, the presence or absence of infection with Streptococcus pyogenes is clinically examined by measuring the amount of ASO in the blood. SLO is used as a raw material for ASO measurement reagents.

[0003] SLO is a protein with a total length of 571 amino acid residues, and the peptide from the N-terminus to the 33rd amino acid residue is a signal peptide. SLO34-571, which does not contain a signal peptide, has been used as a raw material for ASO measurement reagents. In recent years, modified SLOs with deleted C-terminal regions have been developed with the aim of providing SLO with no hemolytic activity and high thermostability (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017 / 204325 Summary of the Invention [Problem to be solved by the invention]

[0005] The position of the epitope of a neutralizing antibody against SLO in SLO has not yet been studied. The present inventors considered that a protein containing an epitope of a neutralizing antibody against SLO can be used as a vaccine antigen capable of producing a neutralizing antibody, and can also be used as a test antigen capable of quantifying a neutralizing antibody titer. That is, an object of the present invention is to determine the position of the epitope of a neutralizing antibody against SLO and to provide a protein (SLO antigen) containing such an epitope. [Means for solving the problem]

[0006] The present inventors investigated the reactivity of neutralizing antibodies against SLO and found that the reactivity was 100% against SLO82-571 and about 70% against SLO34-460 and SLO82-460, and demonstrated that SLO82-460 is the major epitope of neutralizing antibodies against SLO. Based on these findings, the present inventors have completed the present invention.

[0007] That is, the present invention relates to the following [1] to

[21] . [1] A protein selected from the following (a) to (g): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO:3 (b) a protein consisting of the amino acid sequence set forth in SEQ ID NO:2 (c) a protein consisting of the amino acid sequence set forth in SEQ ID NO:4 (d) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which a portion of the amino acid residues at positions 1 to 48 is deleted (e) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 in which a portion of the amino acid residues at positions 380 to 490 is deleted (f) a protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence of any one of the proteins (a) to (e) and that binds to an ASO (g) A protein comprising any one of the proteins (a) to (f) to which a tag has been added at least one of the C-terminus and the N-terminus. [2] A protein selected from the following (a') to (e'): (a') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 (b') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (c') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which a portion of the amino acid residues at positions 1 to 48 has been deleted (d') A protein that has an amino acid sequence that has 90% or more identity with the amino acid sequence of any of the proteins (a') to (c') and binds to ASO. (e') A protein comprising any one of the proteins (a') to (d') having a tag attached to at least one of the C-terminus and the N-terminus. [3] The protein according to [1] or [2], wherein the tag is a His tag. [4] A diagnostic antigen for measuring a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O, comprising the protein according to any one of [1] to [3] above. [5] A hemolytic streptococcal vaccine comprising the protein according to any one of [1] to [3] above. [6] A DNA encoding the protein according to any one of [1] to [3] above. [7] A vector comprising the DNA described in [6] above. [8] A transformant comprising the vector described in [7] above. [9] The transformant according to [8] above, wherein the transformant is selected from bacteria, insect cells, insects, animal cells, animals, plant cells and plants.

[10] A method for producing the protein according to any one of [1] to [3] above, comprising the step of culturing, raising or cultivating the transformant according to [8] or [9] above.

[11] A baculovirus comprising the vector described in [7] above integrated into bacmid DNA.

[12] A method for producing the protein according to any one of [1] to [3] above, comprising the step of infecting a host insect cell or insect with the baculovirus according to

[11] above.

[13] A method for producing the protein according to any one of [1] to [3] above, wherein the vector according to [7] above is a viral vector, the method comprising a step of infecting a host animal cell or animal with the vector.

[14] An Agrobacterium comprising the vector described in [7] above.

[15] A method for producing the protein according to any one of [1] to [3] above, comprising the step of infecting a host plant with the Agrobacterium according to

[14] above.

[16] Latex particles having the protein according to any one of [1] to [3] above adsorbed thereon.

[17] A kit for measuring ASO, comprising the latex particles described in

[16] above.

[18] The kit described in

[17] above, wherein the ASO is a neutralizing antibody that neutralizes the hemolytic activity of SLO.

[19] A method for measuring ASO using the latex particles described in

[16] above.

[20] A step of contacting the latex particles described in

[16] above with a test sample that may contain ASO; and measuring an agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein; A method for measuring an anti-SLO antibody, comprising:

[21] The method according to

[19] or

[20] above, wherein the ASO is a neutralizing antibody that neutralizes the hemolytic activity of SLO. Effect of the Invention

[0008] According to the present invention, it is possible to provide a vaccine antigen capable of producing neutralizing antibodies against SLO, and a protein (SLO antigen) that can be used as a testing antigen capable of quantifying neutralizing antibody titer against SLO. [Brief description of the drawings]

[0009] [Figure 1]1 shows the results of SDS-PAGE of each SLO variant under non-reducing conditions, with the leftmost lane being molecular weight marker, lane 1 being SLO34-460, lane 2 being SLO82-460, and lane 3 being SLO82-571. [Diagram 2] 1 shows the results of an antibody absorption test using SLO34-460, where (a) shows the results when ASO-positive serum before antibody absorption was added as a ligand, and (b) shows the results when the flow-through fraction after antibody absorption was added as a ligand. [Diagram 3] 1 shows the results of an antibody absorption test using SLO82-460, where (a) shows the results when ASO-positive serum before antibody absorption was added as a ligand, and (b) shows the results when the flow-through fraction after antibody absorption was added as a ligand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the present invention will be described in detail.

[0011] One embodiment of the present invention is a protein selected from the following (a) to (g): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, (b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (c) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, (d) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 with a portion of the amino acid residues at positions 1 to 48 deleted, (e) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 with a portion of the amino acid residues at positions 380 to 490 deleted, (f) a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of any of the proteins (a) to (e) and binding to ASO, or (g) a protein having a tag added to at least one of the C-terminus and N-terminus of any of the proteins (a) to (f). Such a protein contains an epitope of a neutralizing antibody against SLO, and therefore can be a vaccine antigen capable of producing a neutralizing antibody.

[0012] SLO34-571, which is obtained by removing amino acid residues at positions 1 to 33 (signal peptide) from full-length SLO, is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1. SLO34-460, which is obtained by removing amino acid residues at positions 1 to 33 and 461 to 571 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 2". SLO82-460, which is obtained by removing amino acid residues at positions 1 to 81 and 461 to 571 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 3". SLO82-571, which is obtained by removing amino acid residues at positions 1 to 81 from full-length SLO, is a "protein consisting of the amino acid sequence shown in SEQ ID NO: 4".

[0013] "A protein consisting of the amino acid sequence of SEQ ID NO:2 in which some of the amino acid residues at positions 1 to 48 have been deleted" corresponds to a protein in which some of the amino acid residues at positions 34 to 81 of full-length SLO have been deleted from SLO34-460. The positions and number of amino acid residues to be deleted are not limited, and any one or more amino acid residues within the range of positions 1 to 48 may be deleted.

[0014] "A protein consisting of the amino acid sequence of SEQ ID NO: 4 in which a portion of the amino acid residues at positions 380 to 490 are deleted" corresponds to a protein in which a portion of the amino acid residues at positions 461 to 571 of full-length SLO is deleted from SLO82-571. The positions and number of amino acid residues to be deleted are not limited, and any one or more amino acid residues within the range of positions 461 to 571 may be deleted.

[0015] The identity of the amino acid sequence in a "protein that has an amino acid sequence that is 90% or more identical to the amino acid sequence of any of proteins (a) to (e) and binds to ASO" may be 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or may be less than 100%. Methods for determining whether a protein binds to an ASO include immunological assays performed on a detection substrate on which the protein is immobilized, such as, but not limited to, ELISA, immunochromatography, immunoagglutination (LA), chemiluminescent immunoassay (CLIA), pulse immunoassay, time-resolved fluorescence resonance energy transfer (TR-FRET), QCM (Quartz Crystal Microbalance), BioLayer Interferometry (BLI), and surface plasmon resonance. It can also be applied to immunological assays that do not involve immobilizing proteins on a detection substrate, such as turbidimetric immunoassay (TIA), time-resolved fluorescence resonance energy transfer (TR-FRET), QCM (Quartz Crystal Microbalance), BioLayer Interferometry (BLI), and surface plasmon resonance, but this is not limited to these. Here, to determine whether or not a protein binds to an ASO, a test statistic calculated from two measurements taken when a protein is applied to a test group (i) that definitely does not contain ASO and a test group (ii) that may contain ASO is used to test the difference in the population means of two populations.The test statistic may be the difference obtained by subtracting the measured value of (i) from the measured value of (ii), or the value obtained by dividing the measured value of (ii) by the measured value of (i), but the test statistic to be adopted is not limited to these. Therefore, the null hypothesis H0:μ is determined using the test statistic. i =μ ii , Alternative hypothesis H1:μ i ≠μ ii When the significance level α is set to 0.05, if the test statistic t0 is in the rejection region, H0 is rejected and it is determined that the protein and ASO have bound to each other.

[0016] The tag in "a protein having a tag added to at least one of the C-terminus and N-terminus of any of the proteins (a) to (f)" is a peptide or protein added for the purpose of isolating, purifying, immobilizing, detecting, solubilizing, visualizing, etc. of the protein. For example, solubility tags include glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (TRX), and Nus tags, and detection tags include S tags, HSV tags, FLAG tags, His tags, Strep tags, Strep-II tags, Myc tags, HA tags, V5 tags, E tags, T7 tags, VSV-G tags, Glu-Glu tags, and Avi tags. His tags, Strep tags, and Strep-II tags can also be used as purification tags. Proteins to which these tags have been added can be expressed as fusion proteins. Proteins to which these tags have been added can be used as they are as vaccine antigens capable of producing neutralizing antibodies. Furthermore, a protein to which these tags have been added may be treated with a digestive enzyme to cleave the tag, thereby obtaining any of the proteins (a) to (f). Among these tags, the His tag is preferred. The His tag can be used for purifying proteins by utilizing its property of specifically binding to metal ions such as nickel. The number of histidine residues contained in the His tag may be, for example, 4 to 10, 5 to 7, or 6.

[0017] The protein is preferably any one of proteins selected from the following (a') to (e'): (a') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3, (b') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, (c') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which a portion of the amino acid residues at positions 1 to 48 have been deleted, (d') a protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of any one of the proteins (a') to (c') and binding to ASO, and (e') a protein having a tag attached to at least one of the C-terminus and N-terminus of any one of the proteins (a') to (d'). Such a protein contains SLO82-460, which is the main epitope of neutralizing antibodies against SLO, and is therefore particularly useful as a vaccine antigen capable of producing neutralizing antibodies.

[0018] One embodiment of the present invention is a diagnostic antigen for measuring a neutralizing antibody that neutralizes the hemolytic activity of SLO, which is composed of the above protein. As described below in the kit for measuring ASO and the method for measuring ASO, the above protein can be used as a diagnostic antigen for measuring the above neutralizing antibody. The above diagnostic antigen can be used to qualitatively evaluate the presence or absence of the above neutralizing antibody in a test sample, or to quantitatively evaluate the concentration of the above neutralizing antibody in a test sample. If the neutralizing antibody ability of a subject can be measured, it may be possible to predict the severity of the subject and the risk of reinfection.

[0019] One embodiment of the present invention is a Streptococcus haemolyticus vaccine comprising the above protein.

[0020] The vaccine may be in the form of, for example, liquid, powder (lyophilized powder, dry powder), capsule, tablet, or frozen state.

[0021] The vaccine may contain a medicamentously acceptable carrier. As the carrier, any carrier commonly used in vaccine production may be used without limitation. Specifically, the carrier may include saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffer, and combinations thereof. The vaccine may further contain an emulsifier, a preservative (e.g., thimerosal), an isotonicity agent, a pH adjuster, an inactivating agent (e.g., formalin), etc., as appropriate.

[0022] The route of administration of the vaccine may be, for example, transdermal, sublingual, ophthalmic, intradermal, intramuscular, oral, enteral, nasal, intravenous, subcutaneous, intraperitoneal, or by inhalation from the mouth to the lungs.

[0023] The vaccine may be administered, for example, by syringe, transdermal patch, microneedle, implantable sustained release device, microneedle-attached syringe, needle-free device, or spray.

[0024] To further enhance the immunogenicity of the vaccine, the vaccine may contain an adjuvant together with the protein, such as aluminum adjuvant or squalene-containing oil-in-water emulsion adjuvant (AS03, MF59, etc.), Toll-like receptor ligands such as CpG and 3-O-deacylated-4'-monophosphoryl lipid A (MPL), saponin-based adjuvants, polymer-based adjuvants such as poly-γ-glutamic acid, and polysaccharides such as chitosan and inulin.

[0025] Examples of mammals that are targets of the vaccine of this embodiment include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, humans, etc. The vaccine of this embodiment may be used for humans, or for children under the age of 5 and elderly people over 65 years of age.

[0026] One embodiment of the present invention is a DNA encoding the above protein. By using such a DNA, the above protein can be produced.

[0027] One embodiment of the present invention is a vector comprising the above-mentioned DNA.

[0028] Examples of the vector include a plasmid, a virus, a cosmid, a lambda phage, and an artificial chromosome. Examples of the virus include a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, and a baculovirus. The vector is preferably an expression vector containing a promoter sequence and the like.

[0029] When the expression host is a eukaryote, the vector preferably comprises a promoter sequence, a ribosome binding sequence, a translation initiation site, a polyadenylation tail, a Kozak sequence, and the like.

[0030] When the expression host is a prokaryote such as a bacterium, the vector preferably has an autonomous replicable vector in the prokaryote and has a promoter sequence, a ribosome binding sequence, a translation initiation site, and the like.

[0031] In addition to these, the vector can also contain, for example, a replication origin, a genetic marker, antibiotic resistance, an epitope, a targeting sequence, and a reporter gene.

[0032] One embodiment of the present invention is a transformant comprising the above-mentioned vector.

[0033] The transformant may be, for example, a prokaryote such as a bacterium, a yeast, a filamentous fungus, an insect, an animal, an algae, a plant, or a cell thereof. The transformant is preferably selected from a bacterium, an insect cell, an insect, an animal cell, an animal, a plant cell, and a plant.

[0034] Examples of bacteria include Escherichia coli, bacteria of the genus Brevibacillus, bacteria of the genus Corynebacterium, and bacteria of the genus Rhodococcus.

[0035] Examples of yeast include yeasts belonging to the genera Saccharomyces, Pichia, Candida, and Torulopsis.

[0036] Examples of filamentous fungi include filamentous fungi belonging to the genera Aspergillus, Penicillium, and Trichoderma.

[0037] Examples of insects include insects of the Noctuidae and Bombycidae families of the Lepidoptera order.

[0038] Examples of animals include mammals, and examples of animal cells include cells derived from human cervical cancer, human fetal kidney, hamster ovary, and African green monkey kidney.

[0039] Examples of algae include algae belonging to the genera Chlamydomonas and Synechococcus.

[0040] Examples of plants include plants belonging to the Solanaceae family (e.g., tobacco, eggplant, tomato, bell pepper, and chili pepper), Rosaceae family (e.g., rose and strawberry), Brassicaceae family (e.g., Arabidopsis thaliana, rapeseed, Chinese cabbage, cabbage, radish, and rapeseed), Asteraceae family (e.g., chrysanthemum, garland chrysanthemum, and lettuce), Chenopodiaceae family (e.g., spinach and sugar beet), Poaceae family (e.g., wheat, rice, barley, and corn), and Fabaceae family (e.g., soybean, adzuki bean, kidney bean, and broad bean). For example, the plant body may be a plant of the Solanaceae family or the Brassicaceae family, a plant of the genus Nicotiana or a plant of the genus Arabidopsis, and may be Nicotiana benthamiana, Nicotiana tabacum, or Arabidopsis thaliana.

[0041] Examples of methods for transforming an expression host include electroporation, a method using calcium ions, a spheroplast method, a protoplast method, a lithium acetate method, a calcium phosphate method, a lipofection method, and a competent cell method. Examples of methods for expressing a heterologous protein using a plant as an expression host include an agroinfiltration method, a plant virus vector method, the magnICON (registered trademark) system, and a particle gun method.

[0042] The vector may be directly transformed into an expression host, or the vector may be transformed into any cell and the cell may be used to transform into an expression host. Examples of such cells include bacteria, yeast, and animal cells. For example, the protein can be expressed in an expression host or an expression host cell by infecting the expression host or the expression host cell with a transformed bacterium.

[0043] When the transformant is a plant, examples of the bacterium into which the vector is introduced include bacteria of the species Agrobacterium, Rhizobium, Sinorhizobium, Mesorhizobium, Phyllobacterium, Ochrobactrum, and Bradyrhizobium, and Agrobacterium is preferred. That is, one embodiment of the present invention is an Agrobacterium containing the above-mentioned vector.

[0044] The case where the plant is a transformant will be described in more detail below. The above protein can be expressed using a plant as an expression host by a known method. Such methods include, for example, the agroinfiltration method, the plant virus vector method, the magnICON (registered trademark) system, and the particle gun method.

[0045] When the agroinfiltration method is used, Agrobacterium is transformed with the above vector, and the Agrobacterium is then infected into a plant, whereby a plant expressing the above protein can be obtained.

[0046] When using the plant virus vector method, a plant expressing the above protein can be obtained as follows. First, RNA is obtained from the cDNA of the plant virus genome into which DNA encoding the above protein has been inserted. Then, this RNA is inoculated as a vector into a plant to infect it. Examples of such virus vectors include tobacco mosaic virus (TMV) vectors, plum pox virus (PPV) vectors, potato virus X (PVX) vectors, alfalfa mosaic virus (AIMV) vectors, cucumber mosaic virus (CMV) vectors, cowpea mosaic virus (CPMV) vectors, and zucchini yellow mosaic virus (ZYMV) vectors.

[0047] When using the magnICON® system, plants expressing the above proteins can be obtained as follows: First, the cDNA of the TMV or PVX genome into which the DNA encoding the above protein has been inserted is introduced into a T-DNA vector, and then the plant is infected with Agrobacterium transformed with the obtained T-DNA vector.

[0048] When the transformant is an insect or insect cell, a baculovirus having the above-mentioned vector incorporated into bacmid DNA can be used. That is, one embodiment of the present invention is a baculovirus having the above-mentioned vector incorporated into bacmid DNA. Bacmid DNA is a shuttle vector that can replicate in an insect or insect cell and E. coli, and is a DNA in which a bacterial artificial chromosome (BAC) sequence is introduced into the genomic DNA of a baculovirus that grows in an insect so that it can replicate autonomously in E. coli. The above-mentioned vector is incorporated into the bacmid DNA of the baculovirus of this embodiment. If the bacmid DNA is purified from E. coli and transfected into an insect cell, it can grow in the insect cell as a baculovirus.

[0049] One embodiment of the present invention is a method for producing the protein, comprising the step of culturing, raising or cultivating the transformant, whereby the protein can be expressed in the transformant.

[0050] The production method of this embodiment may further include a step of extracting the expressed protein. Protein extraction can be performed by known methods, such as physical disruption (homogenizer, bead mill, ultrasonic, French press, etc.), osmotic shock, freeze-thaw, addition of surfactant, and enzyme digestion.

[0051] The production method of this embodiment may further include a step of isolating, purifying, and / or concentrating the extracted protein. The isolation, purification, and / or concentration of the protein can be performed by, for example, affinity, ion exchange, gel filtration, and hydrophobic interaction chromatography.

[0052] One embodiment of the present invention is a method for producing the protein, comprising the step of infecting a host plant with the Agrobacterium.

[0053] The production method of this embodiment may further include a step of cultivating the infected host plant. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and / or concentrating the extracted protein. The method of protein isolation, purification and / or concentration is as described above.

[0054] One embodiment of the present invention is a method for producing the protein, comprising the step of infecting a host insect cell or insect with the baculovirus.

[0055] The production method of this embodiment may further include a step of culturing or raising the infected host insect cell or insect. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and / or concentrating the extracted protein. The method of protein isolation, purification and / or concentration is as described above.

[0056] One embodiment of the present invention is a method for producing the protein, comprising the step of infecting a host animal cell or animal with the vector, the vector being a viral vector, including retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and baculoviruses.

[0057] The production method of this embodiment may further include a step of culturing or raising the infected host animal cell or animal. The production method of this embodiment may further include a step of extracting the expressed protein. The method of protein extraction is as described above. The production method of this embodiment may further include a step of isolating, purifying and / or concentrating the extracted protein. The method of protein isolation, purification and / or concentration is as described above.

[0058] One embodiment of the present invention is a latex particle having the above-mentioned protein adsorbed thereon.

[0059] Examples of materials for latex particles include polystyrene and divinylbenzene. The average particle size of latex particles can be 0.1 μm to 5 μm. The particle size of latex particles can be measured by dynamic light scattering. In this specification, the term "average particle size" refers to the particle size (median diameter) when the integrated value from the small particle size reaches 50% of the total in the volume-based particle size distribution curve obtained by dynamic light scattering.

[0060] The above proteins can be adsorbed onto latex particles by a general physical adsorption method.

[0061] One embodiment of the present invention is a kit for measuring ASO, comprising the latex particles described above.

[0062] A kit for measuring ASO can be used to qualitatively evaluate the presence or absence of ASO in a test sample, or to quantitatively evaluate the concentration of ASO in a test sample.

[0063] The kit for measuring ASO may further include a standard sample of ASO and a buffer solution for diluting the test sample.

[0064] The ASO to be measured is preferably a neutralizing antibody that neutralizes the hemolytic activity of SLO. Since the above protein contains an epitope of a neutralizing antibody against SLO, it is also possible to measure the neutralizing antibody titer.

[0065] One embodiment of the present invention is a method for measuring ASO using the above-mentioned latex particles.

[0066] The ASO measurement method can be performed to qualitatively evaluate the presence or absence of ASO in a test sample, or to quantitatively evaluate the concentration of ASO in a test sample.

[0067] In one embodiment, the measurement method includes a step of contacting the latex particles with a test sample that may contain ASO, and a step of measuring the agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein.

[0068] The latex particles can be brought into contact with the test sample that may contain ASO by mixing the suspension containing the latex particles with the test sample. When the two are brought into contact, the latex particles aggregate due to an interaction between the ASO contained in the test sample and the protein, and the absorbance of the suspension changes. The amount of change in absorbance (endpoint method) or the rate of change (rate method) is measured. The measurement is preferably performed by a turbidimetric method or a colorimetric method. For example, the agglutination reaction of the latex particles is measured by irradiating light in the visible to near-infrared range, usually 300 nm to 1000 nm, preferably 500 nm to 900 nm, from the outside of the cell and detecting the change in absorbance or the change in the intensity of the scattered light.

[0069] The time for carrying out the agglutination reaction may be 1 to 30 minutes, preferably 1 to 10 minutes, but is not limited thereto. The temperature for carrying out the agglutination reaction may be 35°C to 40°C, or may be 36 to 38°C, but is not limited thereto.

[0070] A number of standard samples containing the ASO to be measured at various known concentrations are prepared, and the amount or rate of change in absorbance is measured for each of them using the above method. A calibration curve is drawn by plotting the concentration of ASO in the standard samples on the horizontal axis and the amount or rate of change in the measured absorbance on the vertical axis. The amount or rate of change in absorbance of an unknown test sample is also measured using the same method, and the measurement results are applied to the above calibration curve, allowing the ASO in the test sample to be quantitatively evaluated. In addition, a threshold value for the amount or rate of change in absorbance is set in advance, and it can be qualitatively evaluated that the ASO is present in the test sample when the threshold value is exceeded.

[0071] The test sample is not particularly limited as long as it can contain the target antibody, and examples of the test sample include body fluids such as blood, serum, plasma, urine, stool, saliva, tissue fluid, cerebrospinal fluid, swabs, etc., or dilutions thereof, with blood, serum, plasma, urine, stool, cerebrospinal fluid, or dilutions thereof being preferred.

[0072] The ASO to be measured is preferably a neutralizing antibody that neutralizes the hemolytic activity of SLO. Since the above protein contains an epitope of a neutralizing antibody against SLO, it is also possible to measure the neutralizing antibody titer. EXAMPLES

[0073] Test Example 1. Preparation of SLO variants SLO variants lacking some of the amino acid residues of SLO and having the amino acid sequences shown in SEQ ID NOs: 2 to 4 were prepared by the following method. SEQ ID NO:2: SLO34-460 SEQ ID NO:3: SLO82-460 SEQ ID NO:4: SLO82-571

[0074] 1.1. Expression and purification of SLO variants in plants SLO variants were produced using the plant transient expression system described in Japanese Patent Publication No. 5015012. Genes of SLO variants encoding SEQ ID NOs: 2 to 4 were cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics). After introducing these vectors into Agrobacterium for transformation and cultivation, the mixture of culture fluids was infiltrated into Nicotiana benthamiana leaves and harvested in about one week. The harvested leaves (infected leaves) were frozen and then ground in a mortar. 30 ml of extraction buffer was added to 10 g of ground infected leaves and left on ice for 30 minutes. Then, the mixture was centrifuged at 15,000×g and 4°C for 15 minutes, and the supernatant was collected. The collected supernatant was further centrifuged at 15,000×g and 4°C for 10 minutes, and the supernatant was collected. The supernatant was loaded onto a Ni affinity column (His GraviTrap, Cytiva) under the following conditions, and the eluted fraction was collected. The collected eluted fraction was subjected to buffer exchange using a desalting column (Econo-Pac 10DG Desalting Columns, Bio-Rad Laboratories), and concentrated using an ultrafiltration column (Vivaspin Turbo 15, 10,000 MWCO PES, Sartorius).

[0075] Column volume (CV) = 1 mL Steps: Equilibration / 10CV, Load / 30CV, Wash / 15CV, Elute / 3CVx3 Extraction buffer: 20 mM phosphate buffer pH 7.4, 500 mM sodium chloride, 20 mM imidazole Column equilibration buffer: Same as extraction buffer Elution buffer: 20 mM phosphate buffer pH 7.4, 500 mM sodium chloride, 500 mM imidazole The purification yield of each protein when each SLO variant was purified is shown in Table 1. As shown in Table 1, good expression was observed for all SLO variants. In addition, the yields of SLO34-460 and SLO82-460, which are SLO variants in which domain 4 (consisting of amino acid residues 461-571) of SLO was removed, were 2.5-fold or more higher than that of SLO82-571.

[0076] [Table 1]

[0077] The results of SDS-PAGE of each SLO variant under non-reducing conditions are shown in Figure 1. As shown in Figure 1, each of the SLO variants of interest was detected as a major band, confirming that all of the SLO variants were purified to a high degree of purity.

[0078] Test Example 2. Antibody Absorption Test 2.1. Absorption of antibodies to immobilized SLO variants Each SLO variant was immobilized on an antigen immobilization column (Pierce NHS-Activated Agarose Spin Columns, Thermo Scientific), and an ASO-positive serum control (Immunocucela I-(H) "Seiken" RD, Denka) or an ASO-positive serum sample was added and reacted (antibody absorption) for 16 hours at 4° C. The flow-through fraction after the reaction was collected, and the buffer of the flow-through fraction was replaced with PBS using a desalting column (PD MiniTrap G-25, Cytiva).

[0079] 2.2. Confirmation of antibody absorption to immobilized SLO variants Surface plasmon resonance (SPR) was used to confirm whether ASO was present in the flow-through fraction after antibody absorption. SPR was performed using a Biacore (Biacore 8K, Cytiva) equipped with a sensor chip (Series S Sensor Chip Protein A, Cytiva) on which Protein A was immobilized. When ASO-positive serum before antibody absorption or the flow-through fraction of the SLO variant immobilized column after antibody absorption was added as a ligand and the antibody was allowed to bind to Protein A on the sensor chip, a response of about 3000 RU was obtained as the capture level for both. Furthermore, each SLO variant used for immobilization was added at various concentrations as an analyte. The results when the SLO variant and analyte immobilized on the column were SLO34-460 are shown in Figure 2, and the results when they were SLO82-460 are shown in Figure 3.

[0080] When ASO-positive serum before antibody absorption was added as a ligand, a concentration-dependent binding signal to the analyte was observed (Figures 2(a) and 3(a)). On the other hand, when the flow-through fraction from the SLO variant-immobilized column after antibody absorption was added as a ligand, no binding signal was observed even when the SLO variant was added at a relatively high concentration of 900 nM (Figures 2(b) and 3(b)).

[0081] Here, the area of ​​the immobilized portion on the sensor chip is 1.2 mm 2 (Reference 1) and 1000RU≒1ng / mm 2 (Reference 2, p. 13). Therefore, a capture level response of approximately 3000 RU means that the total amount of IgG captured on the sensor chip is 3.6 ng. If the molecular weight of IgG is 150 kDa, 3.6 ng of IgG is approximately 0.024 pmol. In addition, the unit conversion of an analyte concentration of 900 nM is 0.9 pmol / mm 3 The volume of the reaction site on the sensor chip is 0.06 mm 3(Reference 1), the amount of analyte present in the reaction site volume is 0.054 pmol. Therefore, when the concentration of the analyte, i.e., the SLO variant, is 900 nM, the SLO variant is present in the reaction site of the sensor chip in more than twice the amount of antibody (ASO) against the SLO variant, which is sufficient to detect a binding signal. However, the fact that no binding was observed when the flow-through fraction of the SLO variant-immobilized column was added as a ligand indicates that no ASO was present in the flow-through fraction, and there is no stoichiometric contradiction.

[0082] The step in the sensorgram when the analyte is added is due to the so-called solvent effect caused by the difference in refractive index between the analyte solvent (PBS) and the buffer (0.01 M HEPES, pH 7.4, 0.15 M sodium chloride, 3 mM EDTA, 0.05% Tween 20) (Reference 2, p. 64), which is a self-evident fact in the field of testing.

[0083] From the above results, it was confirmed that the flow-through fraction from the modified SLO immobilized column did not contain any antibody (ASO) that binds to the immobilized modified SLO.

[0084] Test Example 3. Neutralizing antibody titer measurement 3.1. Preincubation of SLO variants with ASO SLO34-571 was dissolved in PBS, and DTT was added to 10 mM DTT, and the SLO variant was activated by incubating at room temperature (25°C) for 5 minutes (Reference 3), and DTT was removed using an ultrafiltration column. Next, 80 μl of a solution of the activated SLO variant in PBS at 0.6 μg / ml and 80 μl of a solution of the ASO positive serum control or the flow-through fraction after antibody absorption, which were serially diluted with PBS, were added to a 96-well U-bottom plate and pre-incubated at 25°C for 1 hour.

[0085] 3.2. Hemolysis test Rabbit defibrinated blood (Kohjin Bio, catalog number 12065305) was washed with PBS to prepare 3% rabbit defibrinated blood. 80 μl of 3% rabbit defibrinated blood was added to the plate on which SLO34-571 and ASO had been preincubated, and incubated at 37°C for 45 minutes. The plate was then centrifuged at 1000xg for 5 minutes, and 200 μl of the supernatant was transferred to a flat-bottom plate for absorbance measurement, and the absorbance at 541 nm, which indicates the degree of hemolysis, was measured (References 4 and 5). The neutralizing ability (%) at each dilution step was calculated from the degree of hemolysis using the following formula. Neutralizing activity (%) = 100 - {(sample - non-hemolytic control) / (hemolytic control - non-hemolytic control)} x 100 Samples: The absorbance (OD) of each sample 541 ) Non-hemolytic control: SLO variant and ASO positive serum control, or absorbance (OD ) when PBS solution was added instead of the flow-through fraction after antibody absorption. 541 ) Hemolysis control: The absorbance (OD) when PBS solution was added instead of the ASO positive serum control or the flow-through fraction after antibody absorption. 541 )

[0086] The dilution ratio showing 50% neutralization was calculated from the neutralization activity (%) at each dilution step (IC 50 The neutralizing antibody titers before and after antibody absorption using the ASO positive serum control are shown in Table 2.

[0087] [Table 2]

[0088] As is clear from the results shown in Table 2, the neutralizing activity of the flow-through fraction when SLO34-460 or SLO82-460 was immobilized was reduced by about 70% compared to the ASO-positive serum control before antibody absorption. Therefore, it can be said that the reactivity of neutralizing antibodies against each SLO variant is as shown in Table 3.

[0089] [Table 3]

[0090] Similar experiments were performed using clinical samples (serum) known to be ASO positive instead of the ASO-positive serum control, and similar results were obtained, as shown in Tables 4 and 5.

[0091] [Table 4]

[0092] [Table 5]

[0093] Similar experiments were also performed on other ASO-positive clinical samples. As is clear from the results shown in Tables 6 and 7, the neutralizing antibody titers (IC 50 Similarly, in different ASO-positive clinical samples, SLO34-460 and SLO82-460 were shown to capture approximately 70% of neutralizing antibodies. The above results show that, among neutralizing antibodies against SLO, the abundance ratio of neutralizing antibodies having SLO34-460 and SLO82-460 as epitopes was constant at about 70% regardless of the sample. In addition, since it is clear from these results that the remaining 30% of neutralizing antibodies are antibodies whose epitope is domain 4 (consisting of amino acid residues 461-571), Tables 6 and 7 do not show the neutralizing activity (%) of the flow-through fraction upon immobilization of SLO82-571 or the reactivity of neutralizing antibodies to SLO82-571.

[0094] [Table 6]

[0095] [Table 7]

[0096] The above results indicate that SLO34-460 and SLO82-460 can capture approximately 70% of neutralizing antibodies regardless of the specimen, and the remaining 30% of neutralizing antibodies have domain 4 as an epitope. Therefore, SLO34-460 and SLO82-460 are not only useful for diagnosing hemolytic streptococcal infections, but are also the major epitopes of neutralizing antibodies that exhibit clinically important protective ability against hemolysis caused by hemolytic streptococcal infections. Therefore, these antigens are considered to be useful as vaccine antigens against hemolytic streptococci.

[0097] References Reference 1: Morio Arai, "Analysis of biomolecular interactions using a biosensor (BIACORE) using surface plasmon resonance - Application to research in the field of thrombosis and hemostasis", Journal of the Japanese Society on Thrombosis and Hemostasis, Vol. 8 (No. 5), pp. 397-405, 1997 Reference 2: Setsuko Hashimoto and Kaori Morimoto (eds.), "Interaction Analysis Using Biacore: Experimental Methods," Maruzen Publishing, 2012 Reference 3: BHAKDI, SUCHARIT, et al., “Isolation and identification of two hemolytic forms of streptolysin-O.”, Infection and immunity, 1984, 46.2: 394-400. Reference 4: DALE, James B., et al., “Antibodies against a synthetic peptide of SagA neutralize the cytolytic activity of streptolysin S from group A streptococci.”, Infection and immunity, 2002, 70.4: 2166-2170. Literature 5: HUGO, FERDINAND, et al., “Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O.”, Infection and immunity, 1986, 54.3: 641-645.

Claims

1. Any protein selected from the following (a) to (c): (a) a protein consisting of the amino acid sequence set forth in SEQ ID NO:3 (b) a protein having an amino acid sequence having 90% or more identity with the amino acid sequence of the protein of (a), which binds to an anti-streptolysin O antibody. (c) A protein comprising any one of the proteins (a) to (b) to which a tag has been added at least one of the C-terminus and the N-terminus.

2. The protein of claim 1 , wherein the tag is a His tag.

3. A diagnostic antigen for measuring a neutralizing antibody which neutralizes the hemolytic activity of streptolysin O, comprising the protein according to claim 1 or 2.

4. A Streptococcus haemolyticus vaccine comprising the protein of claim 1 or 2.

5. A DNA encoding the protein according to claim 1 or 2.

6. A vector comprising the DNA of claim 5.

7. A transformant comprising the vector according to claim 6.

8. The transformant according to claim 7 , wherein the transformant is selected from bacteria, insect cells, insects, animal cells, non-human animals, plant cells and plants.

9. A method for producing the protein according to claim 1 or 2, comprising the steps of: A method comprising a step of culturing, raising or cultivating the transformant according to claim 7 or 8.

10. A baculovirus comprising the vector according to claim 6 integrated into bacmid DNA.

11. A method for producing the protein according to claim 1 or 2, comprising the steps of: A method comprising the step of infecting a host insect cell or insect with the baculovirus of claim 10.

12. A method for producing the protein according to claim 1 or 2, comprising the steps of:

7. A method according to claim 6, wherein the vector is a viral vector, the method comprising a step of infecting a host animal cell or a non-human animal with the vector.

13. An Agrobacterium comprising the vector of claim 6.

14. A method for producing the protein according to claim 1 or 2, comprising the steps of: A method comprising a step of infecting a host plant with the Agrobacterium of claim 13.

15. 3. Latex particles having the protein according to claim 1 or 2 adsorbed thereon.

16. A kit for measuring anti-streptolysin O antibodies, comprising the latex particles according to claim 15.

17. The kit according to claim 16 , wherein the anti-streptolysin O antibody is a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O.

18. A method for measuring anti-streptolysin O antibodies, which uses the latex particles according to claim 15.

19. A step of contacting the latex particles according to claim 15 with a test sample that may contain an anti-streptolysin O antibody; and measuring an agglutination reaction of the latex particles due to an antigen-antibody reaction between the antibody and the protein; A method for measuring an anti-streptolysin O antibody, comprising:

20. The measurement method according to claim 18 or 19, wherein the anti-streptolysin O antibody is a neutralizing antibody that neutralizes the hemolytic activity of streptolysin O.

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