JP5769289B2 - Reagent for testing mycosis caused by Suehirotake - Google Patents

Description

  The present invention relates to a novel test method and test reagent for testing allergic bronchopulmonary mycosis caused by Shirohirotake.

  Schizophyllum commune is a kind of basidiomycete that is ubiquitous in the environment. In the 1950's, this Shirohirotake was recognized as a pathogenic fungus, and several reports have reported that Suehirotake is a causative fungus of humans (Non-Patent Documents 1 to 3). See).

  Since then, many cases of mycosis caused by Shirohirotake have been reported all over the world, especially in developed countries. Suehirotake is now allergic bronchopulmonary mycosis (ABPM), allergic fungal sinusitis (AFS) fungal sinusitis), and a causative bacterium of human mycosis such as pulmonary basidiomycosis (see Non-Patent Document 4).

  Symptoms of patients infected with Suehirotake are not characteristic and it is often difficult to isolate Suehirotake from clinical samples (saliva, BALF, etc.). Furthermore, even if it is possible to isolate Shirohirotake from a clinical sample, its morphological identification is very difficult, so that there is currently not enough established means for making a definitive diagnosis. is there.

  Non-Patent Document 5 discloses an auxiliary diagnosis method for allergic bronchopulmonary mycosis (ABPM) caused by Shirohirotake. Specifically, focusing on the fact that Suehirotake's culture supernatant contains antigenic substances, clinically assisted diagnosis of ABPM, including measuring the antibody titer of patient serum using the culture supernatant The law is disclosed.

  However, the culture supernatant used in the method disclosed in Non-Patent Document 5 contains various proteins in addition to the protein (antigen protein) as the essential form of the antigen. Further, in the method using the culture supernatant, there is a concern that the composition of the culture supernatant varies greatly from one preparation lot to another. Therefore, the technique disclosed in Non-Patent Document 5 has a problem in that reproducibility and quantitative performance are reduced due to an increase in background, occurrence of a cross-reaction, variation in the composition of the culture supernatant, and the like.

  By the way, glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharides and polysaccharide molecules. And is widely used in the food industry (see Non-Patent Document 6). And it is also known that Shirohirotake produces this glucoamylase (refer nonpatent literature 7).

  On the other hand, there have been some reports that glucoamylase produced by fungi other than Shirohirotake can function as an antigen for allergic diseases. For example, it has been reported that patients with certain types of asthma (Baker's asthma) react positively with glucoamylase derived from Aspergillus niger and produce IgE antibodies against glucoamylase (see Non-Patent Document 8). reference). There is also a report that a glucoamylase derived from Penicillium chrysogenum known as so-called blue mold is a foreign antigen (see Non-Patent Document 9). However, there has been no report on the glucoamylase produced by Suehirotake having any antigenicity.

Kligman, A.M .: A Basidiomycete Probably Causing Onychomycosis 1. The. Journal. Of. Investigative. Dermatology. 14: 67-70, 1950 Batista, A.D., Maia, J.A., Singer, R .: Basidioneuromycosis on man. An. Soc. Biol. Pernambuco. 13: 52-60, 1995 Ciferri, R., Batista, A.C., Campos, S .: Isolation of Schizophyllum commune from a sputum. Atti. Ist. Bot. Lab. Crittogam. Univ. Pavia. 14: 118-120, 1956 Kamei, K., Unno, H., Ito, J., Nishimura, K., Miyaji, M .: [Analysis of the cases in which Schizophyllum commune was isolated] Nippon. Ishinkin. Gakkai. Zasshi. 40: 175-181 , 1999 Kamei, K., Unno, H., Nagao, K., Kuriyama, T., Nishimura, K., Miyaji, M .: Allergic bronchopulmonary mycosis caused by the basidiomycetous fungus Schizophyllum commune. Clin. Infect. Dis. 18: 305 -309, 1994 Kumar, P., Satyanarayana, T .: Microbial glucoamylases: characteristics and applications. Crit. Rev. Biotechnol. 29: 225-255, 2009 Ohm, RA, de Jong, JF, Lugones, LG, Aerts, A., Kothe, E., Stajich, JE, de Vries, RP, Record, E., Levasseur, A., Baker, SE, Bartholomew, KA, Coutinho, PM, Erdmann, S., Fowler, TJ, Gathman, AC, Lombard, V., Henrissat, B., Knabe, N., Kues, U., Lilly, WW, Lindquist, E., Lucas, S. , Magnuson, JK, Piumi, F., Raudaskoski, M., Salamov, A., Schmutz, J., Schwarze, FW, Vankuyk, PA, Horton, JS, Grigoriev, IV, Wosten, HA: Genome sequence of the model mushroom Schizophyllum commune. Nat. Biotechnol. 2010. Jul.: 11, 2010 Quirce, S., Fernandez Nieto, M., Bartolome, B., Bombin, C., Cuevas, M., Sastre, J .: Glucoamylase: another fungal enzyme associated with baker's asthma. Ann. Allergy. Asthma. Immunol. 89 : 197-202, 2002 Luo, W., Wilson, A.M., Miller, J.D .: Characterization of a 52 kDa exoantigen of Penicillium chrysogenum and monoclonal antibodies suitable for its detection.Mycopathologia. 169: 15-26, 2010

  In view of the prior art as described above, an object of the present invention is to provide a means for examining mycosis caused by Shirohirotake, which is excellent in reproducibility and quantification.

  The present inventors have conducted intensive research with the aim of solving the above-described problems. As a result, unexpectedly, it was found that the antigenic substance contained in the culture supernatant of Shirohirotake was glucoamylase, and the present invention was completed.

  That is, according to the first aspect of the present invention, there is provided a method for testing mycosis caused by Shirohirotake, which comprises detecting an antibody that specifically recognizes a glucoamylase protein in a biological sample. In the test method, the biological sample is preferably a body fluid, and the body fluid is preferably serum. In the test method, the mycosis is preferably allergic bronchopulmonary mycosis (ABPM).

  Furthermore, according to the 2nd form of this invention, the reagent for the test | inspection of the mycosis by Suehirotake which contains the substance which detects the antibody which recognizes glucoamylase protein specifically is provided. In the test reagent, the “substance that detects an antibody specifically recognizing glucoamylase protein” is preferably a glucoamylase protein and / or a partial peptide thereof, and the glucoamylase protein is preferably a recombinant protein. It is. In the test reagent, the mycosis is preferably allergic bronchopulmonary mycosis (ABPM).

  ADVANTAGE OF THE INVENTION According to this invention, the test | inspection means of the mycosis by Suehirotake which was excellent in reproducibility and quantitative property can be provided.

In the Examples, the results of electrophoresis (SDS-PAGE) of Suehirotake culture supernatant and CBB staining and the results of Western blotting using sera from patients with allergic bronchopulmonary mycosis (ABPM) It is a photograph shown. In an Example, it is a photograph which shows the result confirmed by CBB dyeing | staining after SDS-PAGE that the recombinant glucoamylase protein expressed by colon_bacillus | E._coli shows a single band. In an Example, it is a graph which shows the result of having quantified and compared serum IgG titer by ELISA method between the patient group of allergic bronchopulmonary mycosis (ABPM) and the healthy volunteer group. In an Example, it is a graph which shows the result of having quantified and compared the IgE titer of the serum by ELISA method between the patient group of allergic bronchopulmonary mycosis (ABPM), and a healthy volunteer group.

  A first aspect of the present invention is a method for examining mycosis caused by Shirohirotake, which comprises detecting an antibody that specifically recognizes a glucoamylase protein (hereinafter sometimes referred to as “glucoamylase”) in a biological sample. It is.

  The subject to which the inspection method of the present invention can be applied is not particularly limited as long as it is an animal, and examples thereof include mammals. Mammals include, for example, primates, laboratory animals, livestock, pets and the like, and are not particularly limited. Specifically, for example, humans, monkeys, rats, mice, rabbits, horses, cows , Goats, sheep, dogs, cats and the like. Preferably, the target animal is a human.

  Although it does not specifically limit as a biological sample which can be used for the method of this invention, For example, the tissue derived from the animal which is a test object, a cell, a cell extract component, a body fluid, etc. are mentioned. Tissues include spleen, lymph nodes, kidneys, lungs, heart, liver, etc. Cells include spleen cells, lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, antibody-producing cells, etc. as body fluids Examples include blood, serum, plasma, urine, sweat, spinal fluid and the like. In view of the ease of detection and the like, the biological sample is preferably a body fluid, particularly serum / plasma or urine, and serum is most preferred.

  There is no particular limitation on the specific form of “mycosis caused by Shirohirotake” to be tested in the test method of the present invention, and any disease, symptom, disorder, etc. that can be recognized as “mycosis caused by Shirohirotake” in this technical field. Can also be included. Examples of mycosis caused by Shirohirotake include allergic bronchopulmonary mycosis (ABPM), allergic fungal sinusitis (AFS), basidiomycosis of the lung, brain abscess, bronchus Examples include, but are not limited to, mucoid impaction of bronchi. However, the test method of the present invention is preferably a test method for allergic bronchopulmonary mycosis (ABPM) among mycoses caused by Shirohirotake.

  The glucoamylase recognized by the antibody (hereinafter sometimes referred to as “anti-glucoamylase antibody”) detected in the test method of the present invention is 1,4-α-D-glucan glucohydrolase (EC 3.2.1. 3) also referred to as an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. The glucoamylase is not particularly limited as long as it is derived from Shirohirotake. As the glucoamylase used in the present invention, for example, natural glucoamylase derived from Suehirotake can be used. The amino acid sequence of glucoamylase is known. For example, glucoamylase derived from Shirohirotake can refer to the database of each public institution based on the following ID number: SCHCODRAFT_57589 glycoside hydrolase family 15 and carbohydrate-binding module family 20 [Schizophyllum commune H4-8] Gene ID: 9591574.

As glucoamylase,
(A) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence of a natural glucoamylase, and having the biological activity of glucoamylase; or ( b) It consists of an amino acid sequence having a homology of 90% or more, preferably 93% or more, more preferably 95% or more, and even more preferably 99% or more with the amino acid sequence of natural glucoamylase, and the biology of glucoamylase Protein with active activity,
Can also be used in the present invention. These glucoamylases can be produced by any genetic engineering technique known to those skilled in the art.

  “Homology” (or “identity”) means that the two amino acid residues constituting the chain are identical in the mutual compatibility between the two chains in the amino acid sequence of the polypeptide. It means the amount (number) of things that can be determined to be, and means the degree of sequence correlation between two polypeptide sequences or two polynucleotide sequences. This homology can be easily calculated. Many methods are known for measuring homology between two polynucleotide or polypeptide sequences, and the term “homology” is well known to those skilled in the art (eg, Lesk, AM (Ed.), Computational Molecular Biology, Oxford University Press, New York, (1988); Smith, DW (Ed.), Biocomputing: Informatics and Genome Projects, Academic Press, New York, (1993); Grifin, AM & Grifin, HG (Ed. ), Computer Analysis of Sequence Data: Part I, Human Press, New Jersey, (1994); von Heinje, G., Sequence Analysis in Molecular Biology, Academic Press, New York, (1987); Gribskov, M. & Devereux, J. (Ed.), Sequence Analysis Primer, M-Stockton Press, New York, (1991), etc.). Common methods used to measure the homology of two sequences include Martin, J. Bishop (Ed.), Guide to Huge Computers, Academic Press, San Diego, (1994); Carillo, H. & Lipman , D., SIAM J. Applied Math., 48: 1073 (1988), etc., but are not limited thereto.

The class of the antibody is not particularly limited, and may be any of IgG, IgD, IgE, IgA, sIgA, IgM and the like. IgG or IgE is preferable, and IgG is particularly preferable. Antibody binding fragments (Fab, Fab ′, F (ab ′) _2, etc.) are also included in the “antibody” as long as they specifically bind to glucoamylase.

  As a method for detecting “an antibody specifically recognizing glucoamylase” in a biological sample, a method known per se can be used, and it is not particularly limited, but a reaction that occurs in a liquid phase or a solid phase (for example, an antigen-antibody reaction). ) Directly, or a method of measuring inhibition of an immune reaction by adding an inhibitor.

  Examples of the method include a method in which glucoamylase or a partial peptide thereof is brought into contact with a biological sample, and specific binding of an antibody in the biological sample to glucoamylase or a partial peptide thereof is detected directly or indirectly. .

  In the present invention, the partial peptide of glucoamylase is not particularly limited as long as it is a partial peptide containing an antigenic determinant recognized by the anti-glucoamylase antibody detected in the present invention. In general, since an antigenic determinant of a protein antigen is composed of at least 5 to 6 amino acid residues, a glucose containing at least 5 or more, preferably 8 or more, more preferably 10 or more amino acid residues. A partial peptide of amylase can be used in the present invention.

  Glucoamylase or a partial peptide thereof may be modified. Examples of such modifications include modifications with phosphoric acid, sugars or sugar chains, phospholipids, lipids, nucleotides, and the like.

  The glucoamylase or its partial peptide used in the present invention can be obtained from Suhirotake, for example, by a method known per se. For example, glucoamylase or a partial peptide thereof can be purified from Suehirotake by a method such as separation from Suehirotake crushed cells or culture supernatant using various column chromatography.

  The glucoamylase of the present invention or a partial peptide thereof is a culture obtained by culturing a transformant introduced with an expression vector containing a nucleic acid encoding glucoamylase or a partial peptide thereof to produce glucoamylase or a partial peptide thereof. It can also be produced by separating and purifying glucoamylase or its partial peptide from

  The glucoamylase or its partial peptide used in the present invention can also be produced by a known peptide synthesis method. Such a peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. A glucoamylase or a partial peptide thereof can be produced by condensing a partial peptide or amino acid capable of constituting glucoamylase and the remaining portion, and removing the protective group when the product has a protective group.

  The partial peptide of glucoamylase used in the present invention can also be produced by cleaving glucoamylase obtained by any of the methods described above or below with an appropriate peptidase.

  The glucoamylase or its partial peptide may be one to which an appropriate tag is linked for the purpose of facilitating the purification operation or the like. Examples of such tags include an immunoglobulin Fc region, maltose binding protein (MBP), glutathione-S-transferase (GST), c-Myc tag, FLAG tag, HA tag, His tag and the like.

Although it does not specifically limit as a detection method for detecting an antibody, More specifically, the following method is mentioned.
(1) An agglutination reaction in which glucoamylase or a partial peptide (antigen) thereof is coated on the surface of blood cells or gelatin particles, and an antigen-antibody reaction is caused by adding a biological sample to form an aggregate.
(2) A double immune diffusion method (DID: double immune diffusion) in which an extract containing glucoamylase or a partial peptide thereof and a biological sample are diffused in an agar gel to cause a precipitation reaction;
(3) After immobilizing purified glucoamylase or its partial peptide on a plate and adding a biological sample to react,
i) ELISA method in which a secondary antibody conjugated with an enzyme is further reacted to detect color development of the substrate with a spectrophotometer;
ii) a fluorescence immunoassay (FIA) in which a secondary antibody conjugated with a fluorescent dye is further reacted to measure fluorescence development; or
iii) a chemiluminescence immunoassay (CLIA) in which a chemiluminescent substance is further reacted with a secondary antibody conjugated with a chemiluminescent substance to measure chemiluminescence;
(4) An immunoturbidimetric method in which the surface of latex particles, glass beads, or the like is coated with glucoamylase or a partial peptide thereof, light is applied to an agglutination reaction solution that occurs when the particles encounter an antibody, and the transmitted light is measured. Or immuno-ratio method (nephrometry method) to measure the scattered light;
(5) A radioimmunoassay in which glucoamylase or a partial peptide thereof is labeled with a radioisotope and reacted with a biological sample to detect an antigen-antibody reaction;
(6) A frozen thin section or cell of a tissue containing glucoamylase or a partial peptide thereof is attached to a slide glass, and a biological sample is dropped on the section to cause a reaction, which further reacts with a secondary antibody bound to a fluorescent dye. A fluorescent antibody method for detecting fluorescence under a microscope;
(7) A surface plasmon resonance analysis method for checking affinity by immobilizing glucoamylase or its partial peptide on a chip and flowing a biological sample;
(8) Western blotting method in which glucoamylase or a partial peptide thereof in a gel separated and developed by electrophoresis is transferred to a nitrocellulose membrane and reacted with a biological sample to detect an antigen-antibody reaction.

  For example, when the detection means is an ELISA method, specifically, detection and / or quantification can be performed as described below (see Examples described later). That is, according to a conventional ELISA method, for example, a biological sample is provided to each well of a multiwell plate coated with glucoamylase or a partial peptide thereof, and an enzyme-labeled secondary antibody is added to each well and reacted, and an enzyme substrate After adding, the antigen-antibody reaction can be detected and / or quantified by detecting and / or quantifying the product produced by the enzyme.

  In the case of the above-described ELISA method, the enzyme used for labeling may be any conventional enzyme commonly used in ELISA methods. For example, peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, luciferase, esterase, β -D-glucuronidase etc. are mentioned. Peroxidase or alkaline phosphatase can be used publicly in view of being able to achieve more sensitive and stable detection. The enzyme substrate can be appropriately selected depending on the enzyme used. For example, 3,3 ′, 5,5′-tetramethylbenzidine or the like is used in the case of peroxidase, and paranitrophenyl phosphate in the case of alkaline phosphatase. Sodium or the like is used.

  Detection and / or quantification of the product produced by the enzyme can be performed by measuring the absorbance of the product. For example, when 3,3 ′, 5,5′-tetramethylbenzidine is used as the enzyme substrate, the absorbance at 655 nm may be measured.

  For example, when the detection means is a fluorescence immunoassay (FIA), examples of the fluorescent dye include FITC (Fluorescein Isothiocyanate), PE (phycoerythrin), APC (Allophycocyanin), Cy-3, and Cy-5.

  For example, when the detection means is a chemiluminescence immunoassay (CLIA), examples of chemiluminescence include acridinium ester.

  When an antibody that specifically recognizes glucoamylase is detected in a biological sample, it can be determined that the subject from which the biological sample is derived has a high possibility of developing / causing mycosis due to Shirohirotake. In this case, the higher the antibody titer that specifically recognizes glucoamylase in a biological sample, the higher the possibility of developing / causing mycosis by Suehirotake. Conversely, when an antibody specifically recognizing glucoamylase is not detected in a biological sample, it may be determined that the subject from which the biological sample is derived has a low possibility of developing / causing mycosis by Suehirotake. it can.

  When judging the possibility of onset as described above, the judgment criteria are not limited to detection / non-detection of antibodies. For example, an average value ± 3SD of the amount of antibody specifically recognizing glucoamylase in a biological sample derived from a healthy subject is set as a cut-off value, and if the cut-off value is exceeded, the subject develops mycosis by Suehirotake It may be determined that there is a high possibility that the subject is doing, and conversely, if it is equal to or lower than the cut-off value, the subject may be judged to have a low possibility of developing mycosis caused by Shirohirotake.

  Moreover, according to the 2nd form of this invention, the reagent for a test | inspection of an angiitis including the substance which detects the antibody which recognizes the glucoamylase protein in a biological sample specifically is provided.

  The “substance” contained in the test reagent of the present embodiment is not particularly limited as long as it can achieve detection of an antibody that specifically recognizes glucoamylase in the above-described method, but preferably glucoamylase or its It is a partial peptide. In addition, when the anti-glucoamylase antibody detected in the present invention is an antibody group that recognizes a plurality of antigenic determinants, the antibodies specifically recognizing each of the many antigenic determinants present in glucoamylase are covered. From the viewpoint of improving detection sensitivity by detecting the target, the substance is preferably glucoamylase (full length of protein).

  Glucoamylase or a partial peptide thereof may be provided in the form of powder, solution, etc., and blood cells, gelatin particles, plates, latex particles, glass beads, glass slides, chips, microtiter plates, centrifuge tubes, microbeads, membranes Alternatively, it may be provided in a form supported on an insoluble carrier such as a paper disk. In the carrier on the container, glucoamylase or a partial peptide thereof is carried at the site where the solution held on the carrier comes into contact, for example, in the case of a microtiter plate, at the site of the well. The glucoamylase or its partial peptide can be supported on an insoluble carrier by a known method.

  If the reagent for a test | inspection of this invention is used, the mycosis by Suehirotake can be test | inspected easily by the above-mentioned method.

  The test reagent of the present invention can also be a test kit for testing mycosis caused by Shirohirotake, which further includes reagents used in the above-described detection method. Specific examples of the reagent include a buffer for diluting the reagent and biological sample, a fluorescent dye, a reaction vessel, a positive control, a negative control, and an instruction document describing a test protocol. These elements can be mixed in advance if necessary. By using this kit, the examination of mycosis by Suehirotake of the present invention becomes simple, and it is very useful for early treatment policy determination.

  EXAMPLES Hereinafter, although this invention is explained in full detail using an Example, this invention is not limited to a following example.

≪Fungus, cells, growth conditions, serum≫
In this example, IFM47458 (obtained from the Center for Fungal Medicine, Chiba University), a binuclear strain of Shirohirotake, was used as the fungus. This strain is usually cultured at 25 ° C. on potato dextrose agar (PDA) medium.

  In this example, Escherichia coli DH5 strain was used as a cell line for use in production of recombinant protein. This Escherichia coli DH5 strain is usually cultured in L medium in the presence / absence of an appropriate antibiotic.

  In addition, in using human serum in this example, approval was obtained from the Ethics Committee of the Research Center for Fungal Medicine, Chiba University, and informed consent was obtained from patients who collected each serum or from healthy volunteers.

≪Preparation of culture filtrate≫
Inoculate Shirohirotake IFM47458 strain in liquid medium (YNBD) containing 0.5% yeast nitrogen base (Yeast nitrogen base w / o amino acids, BD, Franklin Lakes, New Jersey, USA) and 1% glucose at 35 ° C. For 5 weeks. After the culture medium was filtered, the culture supernatant was subjected to protein precipitation with trichloroacetic acid and acetone. The resulting precipitate was dissolved in a buffer and used for SDS-PAGE.

≪Western blot≫
The protein developed on the gel by SDS-PAGE is visualized (stained) using Coomassie Brilliant Blue R-250 (CBB) (Quick-CDD, Wako Pure Chemical Industries, Ltd., Osaka), or polyvinylidene fluoride Transferred onto a (PVDF) membrane. Thereafter, immunoblot analysis was performed using serum from patients with allergic bronchopulmonary mycosis (ABPM) according to a conventional method. The results of CBB staining and Western blot are shown in FIG. In FIG. 1, “M” lanes indicate molecular weight markers, “SDS-PAGE” lanes indicate the results of CBB staining, and “Western blot” lanes indicate the results of Western blotting.

  As shown in FIG. 1, several proteins were detected by CBB staining, and two strong bands were confirmed by Western blot. Attempts were made to cut out the bands corresponding to these two bands from the gel, but it was difficult to cut them out separately, so these were cut out together and used for mass spectrometry described later.

≪Mass Spectrometry≫
The protein band reacted with the antibody in the patient's serum was excised from the gel after CBB staining, digested with trypsin, and subjected to MS / MS measurement with a mass spectrometer (LTQ XL, Thermo Fisher Scientific). The measurement data was subjected to SEQEST search to identify the protein. The database used was S.commune.allModels.proteins.fasta (January 27, 2009) downloaded from http://genome.jgi-psf.org/Schco1/Schco1.download.ftp.html. We also searched for MASCOT, a database search engine different from SEQEST. At this time, NCBInr_Fungi (January 21, 2011) was used as the database. Decoy database search was performed for both SEQUEST search and MASCOT search, and the condition for identification was that false discovery rate (FDR) was less than 1%. The results are shown in Table 1 below.

  As shown in Table 1, a plurality of proteins were identified from the target band. However, there are significant differences in the MS / MS spectra used for score, sequence coverage, and assignment between the first candidate protein and the second candidate protein, so the desired band has glycoside hydrolase family 15 and carbohydrate- The binding module family 20 is thought to be the most abundant, and this protein is presumed to be glucoamylase from the overall homology. The same protein was also identified using two different search engines. The sequence coverage has different values. This is a difference in whether or not the N-terminal signal sequence of sequences registered in the database is included, and the sequence other than the signal sequence is the same. From the above, it was shown that the target protein is glycoside hydrolase family 15 and carbohydrate-binding module family 20 (namely, glucoamylase).

≪Cloning of glucoamylase gene and expression of glucoamylase in E. coli≫
Based on the known nucleotide sequence of the glucoamylase gene (SCHCODRAFT_57589 glycoside hydrolase family 15 and carbohydrate-binding module family 20 [Schizophyllum commune H4-8] Gene ID: 9591574), the codon usage is optimized and optimized using GeneOptimizer The later gene was synthesized by outsourcing (GENEART AG, Regensburg, Germany) (SEQ ID NO: 1). An Escherichia coli DH5 strain was transformed with this gene cloned into pQE-80L, and the His tag protein was expressed as a recombinant protein. The amino acid sequence of the obtained recombinant protein is shown in SEQ ID NO: 2.

  Specifically, first, the transformed E. coli DH5 strain was cultured at 25 ° C. for 24 hours. After culturing, the strain was suspended in buffer A (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5) and disrupted with a sonicator (BIORUPTOR, Cosmo Bio Inc., Tokyo). The lysate was separated by centrifugation and the pellet containing the inclusion bodies of the recombinant protein was washed with buffer A and gently suspended in buffer A supplemented with 8M urea. The resulting suspension is separated by centrifugation and the supernatant is collected in a slurry (including beads) of Ni Sepharose 6 Fast Flow (GE Healthcare UK Ltd., Buckinghamshire, UK) for the purpose of purifying the His tag protein. Moved to. After allowing the binding to proceed for 15 minutes at room temperature, the slurry was washed with buffer A supplemented with 8M urea, and then buffer B supplemented with 8M urea (50 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole). , PH 7.5). The protein purified in this way was used for further analysis. In addition, it was confirmed by CBB staining after SDS-PAGE that a single band was obtained by purification (FIG. 2).

≪Quantification of serum IgG / IgE titer against recombinant glucoamylase by ELISA≫
ELISA was performed by the following method to quantify the titer of human serum against recombinant glucoamylase.

  200 ng of recombinant glucoamylase was diluted with 200 μL of 0.05M carbonate buffer (pH 9.6) and coated onto the wells of Nunc-immuno plate MaxiSorp (ThermoFischer Scientific Inc., Waltham, Mass., USA). After washing 3 times with TBS, the wells were blocked for 1 hour at 25 ° C. using Protein-Free Blocking Buffer. Using an ImmunoWash Model 1575 (Bio-Rad Laboratories), the wells were washed 3 times with TBS (TBS-T) supplemented with 0.1% Tween-20, and then 1% human serum (allergic bronchopulmonary fungi with Suehirotake) Incubation for 2 hours at 25 ° C. with TBS-T containing the disease (ABPM) patients or healthy volunteers). Thereafter, the wells were washed three times with TBS-T, and goat anti-human IgG antibody (MP Biomedicals, LLC, Solon, Ohio, USA) diluted with TBS-T was added thereto. The plate was incubated at 37 ° C. for 1 hour and washed 3 times with TBS-T. Detection was performed using HRP substrate (TMB Peroxidase EIA Substrate Kit, Bio-Rad Laboratories) according to the instructions in the instructions. After the reaction, the absorbance of the plate at 450 nm was read using Sunrise Rainbow Thermo (Tecan Group Ltd., Mannedorf, Switzerland). At this time, the absorbance of wells not coated with protein was subtracted from the absorbance of coated wells. Data from the patient group (n = 13) and data from the healthy volunteer group (n = 20) were analyzed by Mann-Whitney U test. The results are shown in FIG.

  As shown in FIG. 3, the mean ± standard deviation of IgG titers in the healthy volunteer group was 0.1470 ± 0.0743. On the other hand, the mean value ± standard deviation of IgG titers in the patient group was 1.235 ± 0.6318, showing a significantly high value at a significance level of 1%. This indicates that the recombinant glucoamylase protein also retains antigenicity and can specifically react with patient serum.

  On the other hand, a similar ELISA except that a biotin-conjugated mouse anti-human IgE antibody (clone No. f0822, manufactured by Biomatrix Research, Inc.) and streptavidin-HRP (ThermoFischer Scientific Inc.) were used instead of the anti-IgG antibody. The IgE titer of serum was quantified by the method. In this case, the incubation was performed at 25 ° C. for 1 hour. The results are shown in FIG.

  As shown in FIG. 4, the mean ± standard deviation of IgE titers in the healthy volunteer group was 0.04425 ± 0.07937. In contrast, the mean ± standard deviation of IgE titers in the patient group was 1.812 ± 1.118, which was significantly high at a significance level of 1%.

  From the results shown above, it is suggested that IgG and IgE specific for glucoamylase are produced in most ABPM patients due to Suehirotake. Therefore, according to the present invention, a means (test method, test) for testing mycosis (eg, ABPM) caused by Shirohirotake by detecting an antibody that specifically recognizes a glucoamylase protein in a biological sample. Reagent) may be provided.

  According to the present invention, unlike conventional crude antigens (culture supernatants), a means for testing using a single protein as an antigen has been established. Therefore, the occurrence of problems such as increased background, occurrence of cross-reactions, and fluctuations in the composition of the culture supernatant in the conventional technique using the crude antigen (culture supernatant) is suppressed (preferably prevented), and reproducibility and quantification are achieved. There exists an advantage that the test | inspection means excellent in property is provided.

[SEQ ID NO: 1]
The base sequence of the glucoamylase gene synthesize | combined in the Example is represented.
[SEQ ID NO: 2]
This represents the amino acid sequence of the protein encoded by the gene of SEQ ID NO: 1.

Claims (4)

  A reagent for testing mycosis caused by Shirohirotake, comprising a substance that detects an antibody that specifically recognizes a glucoamylase protein derived from Shirohirotake. The substance is a glucoamylase protein and / or its partial peptide derived from Schizophyllum commune, testing reagent according to claim 1. The reagent for a test | inspection of the allergic bronchopulmonary mycosis by Suehirotake of Claim 2 whose said glucoamylase protein is a recombinant protein. The test reagent according to any one of claims 1 to 3 , wherein the mycosis is allergic bronchopulmonary mycosis.