WO2003072782A1 - Antigene de l'echinocoque, gene codant pour ce dernier et methode permettant de diagnostiquer l'hydatidose humaine - Google Patents

Antigene de l'echinocoque, gene codant pour ce dernier et methode permettant de diagnostiquer l'hydatidose humaine Download PDF

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Publication number
WO2003072782A1
WO2003072782A1 PCT/JP2002/005094 JP0205094W WO03072782A1 WO 2003072782 A1 WO2003072782 A1 WO 2003072782A1 JP 0205094 W JP0205094 W JP 0205094W WO 03072782 A1 WO03072782 A1 WO 03072782A1
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WIPO (PCT)
Prior art keywords
protein
measurement method
antigen
polycysticercosis
actin
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PCT/JP2002/005094
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English (en)
Japanese (ja)
Inventor
Osamu Fujita
Tomoyoshi Nozaki
Original Assignee
Japan Science And Technology Agency
Japan As Represented By Director-General Of National Institute Of Infectious Diseases
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Application filed by Japan Science And Technology Agency, Japan As Represented By Director-General Of National Institute Of Infectious Diseases filed Critical Japan Science And Technology Agency
Publication of WO2003072782A1 publication Critical patent/WO2003072782A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4355—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells

Definitions

  • Multi-hydatid antigen Gene encoding the same, and method for diagnosing polycysticercosis
  • the present invention relates to a novel polyhydatid antigen protein and a gene encoding the same, an immunoassay for detecting an antibody against a polypeptide comprising the antigen protein or a part thereof, and a method for diagnosing human polycysticercosis .
  • Polycysticercosis is also found in almost all areas of Hokkaido and in Aomori Prefecture in Japan below 40 degrees north latitude, and is a multivesicular tapeworm that has foxes, dogs, cats, raccoon dogs, etc.
  • the parasitic sites of polycystic worms are mainly the liver, sometimes the lungs and the brain.
  • Polycysticercosis causes liver dysfunction, etc. It is a highly lethal disease classified as a class of infectious diseases.
  • the condition is asymptomatic for as long as 10 years, and blood tests are often considered to be normal.In advanced stages more than 10 years after infection, hepatomegaly and discomfort in the ribs are found. Hepatoma is remarkable at the end stage, hepatic dysfunction progresses, ascites and portal hypertension symptoms, jaundice, and cachexia are seen, and the patient becomes critically ill with hepatic coma.
  • Methods for diagnosing polycysticercosis include interviews, immunological diagnosis, simple abdominal imaging, ultrasonography, and CT scan.
  • the conventional immunological diagnosis is that There is an ELISA method using a mixed antigen of m2 (glycoprotein) and cytovillin, a recombinant protein.
  • m2 glycoprotein
  • cytovillin a recombinant protein.
  • the diagnostic method is highly sensitive, cross-reaction with closely related species is considerable (approximately 25% with monocysticercosis), and is expensive due to the need for a protein with a higher degree of purification.
  • Primary screening and confirmation of infected individuals Exclusion Not suitable for diagnosis.
  • the inventor of the present invention aimed at providing a test method and a diagnostic method for polycysticercosis, which are excellent in sensitivity and specificity, and which are advantageous in terms of cost by using genetic recombination technology.
  • the present inventors have found a novel protein derived from a polycaptor and a gene encoding the protein, and have completed the present invention. That is, the present invention relates to the following embodiments.
  • Amino acid sequence A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in (a), and which has a fungal actin-regulating activity.
  • Recombinant expression vehicle containing the DNA described in any one of 1 to 3 above (Vehicle)
  • the recombinant expression vehicle must be prepared by any genetic engineering technique known to those skilled in the art.
  • the types are, for example, various plasmid better And expression vectors such as virus vectors, cosmids, and phages.
  • the recombinant expression vehicle according to the above 4 which is an expression vector.
  • the recombinant expression vehicle according to the above item 5 which is an expression vector derived from pBR322.
  • a transformant which is a prokaryotic cell such as a bacterium and various eukaryotic cells such as a yeast cell and a mammalian cell, which have been transformed with the expression vehicle according to 4, 5, or 6.
  • a multiacting insect-derived actin regulatory protein particularly a recombinant protein.
  • a polypeptide comprising a part of the protein according to the above 10 or 11.
  • a diagnostic reagent for polyhydatid disease comprising the protein of 10 or 11 above as an active ingredient.
  • a diagnostic reagent for polyhydatid disease comprising the polypeptide of 12 above as an active ingredient.
  • This kit generally includes test kits such as reaction plates, various buffers, diluents, washing solutions, blocking solutions, secondary reagents such as various enzyme-linked sera, and reaction reagents (color-forming compounds). It can be appropriately selected from other optional components known to those skilled in the art based on the reaction principle used for diagnosis.
  • An immunological measurement method comprising detecting an antibody against the protein according to 9 or 10 in a subject using the polypeptide according to 11 or 12 as an antigen.
  • a method for diagnosing a polycysticercosis patient comprising detecting an antibody against the protein in a body fluid of a subject using the protein according to 9 or 10 above as an antigen.
  • FIG. 1 shows the results obtained by a measurement system using the ELISA method for the antigen Em crude antigen.
  • FIG. 2 shows the results obtained with the ELISA-based assay system for antigen-recombinant cytovirin.
  • FIG. 3 shows the results obtained by the ELISA system for the antigen-recombinant daltathione S-transferase.
  • FIG. 4 shows the results obtained for the antigen-recombinant actin regulatory protein in a measurement system by the ELISA method.
  • the DNA, protein and the like according to the present invention can be obtained and prepared using techniques well known in the art.
  • the immunological measurement method of the present invention can also be performed based on various principles and techniques known to those skilled in the art.
  • Example 1 (Acquisition of DNA encoding actin regulatory protein derived from polycystic insects ⁇ Determination of base sequence)
  • RNAqueous Kit (Ambion, USA) according to the conventional method.
  • the cDNA was synthesized using the ZAP Express cDNA Gigapack III Gold Cloning Kit (Strata gene, USA) according to the conventional method. Synthesized from the previously extracted total RNA
  • the cDNA was introduced into the Xho I site of the ZAP Express vector (pBK-CMV phagemid vector) (Xho I; New England Biolabs, USA), and these were introduced into the E. coli XL1-B1ue MR F ' Conversion was made.
  • Rasmid DNA was purified from plaques that showed a positive reaction using Plasmid Mini Kit (QIAGEN, Germany). The DNA sequence of the inserted fragment was determined using Applied Biosystems ABI Prism 300 (Applied Biosystems, USA). The obtained DNA sequence was analyzed using the Sequence Information Survey (BLAST) of the National Center for Biotechnology Information (NCBI). As a result, we were able to obtain a total of eight types of cDNA, including the three newly discovered types.
  • BLAST Sequence Information Survey
  • NCBI National Center for Biotechnology Information
  • the E. coli cells synthesized with the GST fusion protein were sonicated and passed through a Gluathione Sepharose 4B column (Amersham Pharmacia Biotech, UK) to adsorb only the GST fusion protein onto the column. 0.1% Triton ⁇ After washing the column with PBS. Recombinant protein fraction eluted with Thrombin (Amersham Pharmacia Biotech, UK), Experimental example 3 (ELISA kit preparation and detection)
  • GST glutathion S-transferase-se
  • FIGS 1 to 4 show the results obtained for each antigen.
  • a test method, a diagnostic method, and the like which are excellent in sensitivity and specificity and are economically excellent, are provided.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une méthode d'examen, une méthode de diagnostic, etc., qui se rapportent à l'hydatidose, lesquelles méthodes présentent une sensibilité et une spécificité remarquables et sont avantageuses sur le plan économique en raison de l'utilisation de techniques de recombinaison génétiques. L'invention concerne également un gène codant pour une protéine actine modulaire (AMP) issue de l'échinocoque, une méthode de dosage immunologique permettant de détecter un anticorps dirigé contre un polypeptide comprenant cette protéine antigénique ou une partie de cette dernière, enfin une méthode permettant de diagnostiquer l'hydatidose humaine.
PCT/JP2002/005094 2002-02-27 2002-05-27 Antigene de l'echinocoque, gene codant pour ce dernier et methode permettant de diagnostiquer l'hydatidose humaine WO2003072782A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-52151 2002-02-27
JP2002052151A JP2003250555A (ja) 2002-02-27 2002-02-27 多包虫抗原およびそれをコードする遺伝子およびヒト多包虫症の診断方法

Publications (1)

Publication Number Publication Date
WO2003072782A1 true WO2003072782A1 (fr) 2003-09-04

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JP (1) JP2003250555A (fr)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101949926A (zh) * 2010-08-04 2011-01-19 浙江大学 人体包虫病胶体金免疫层析试尿液快速诊断试纸卡
CN107884578A (zh) * 2017-11-01 2018-04-06 杭州微瑞科技有限公司 用于快速定量检测血清中包虫病抗体检测卡
CN109239211A (zh) * 2018-09-11 2019-01-18 江苏省血吸虫病防治研究所 用于鉴别人体感染包虫的血清标志物及检测试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110716035B (zh) * 2018-07-12 2023-02-28 中国疾病预防控制中心寄生虫病预防控制所 基于棘球蚴微管蛋白为靶点的抗棘球蚴病高通量药物筛选方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
C.P.J. Maury et al., "Finnish hereditary amyloidosis, Amino acid sequence homology between the amyloid fibril protein and human plasma gelsoline", FEBS Lett., 1990, Vol. 260, No. 1, pages 85 to 87 *
Carl W. Dieffenbach et al., "Cloning of Murine Gelsoin and Its Regulation during Differentiation of Embryonal Carcinoma Cells", The Journal Of Biological Chemistry, 1989, Vol. 264, No. 22, pages 13281 - 13288 *
Edward K. Koepf et al., "Equus caballus gelsolin cDNA sequence and protein structural implications", Eur.J.Biochem, 1998, Vol. 251, pages 613 - 621 *
Elisabeth Andre et al., "Severin, Gelsolin, and Villin Share a Homologous Sequence in Regions Presumed to Contain F-actin Severing Domains", The Journal of Biological Chemistry, 1988, Vol. 263, No. 2, pages 722 to 727 *
Elizabeth Cortez-Herrera et al., "Echinococcus garanulosus: Cloning and Functional in Vitro Characterization of an Actin Filament Fragmenting Protein", Experimental Parasitology, 2001, Vol. 97, pages 215 to 225 *
Ossi Turunen et al., "Ezrin has a COOH-Terminal Actinbinding Site that is Conserved in the Ezrin Protein Family", The Journal of Cell Biology, 1994, Vol. 126, No. 6, pages 1445 - 1453 *
Petra M. Frosch et al., "Cloning and characterization of an immunodominant major surface antigen of Echinococcus multilocularis", Molecular and Biochemical Parasitology, 1991, Vol. 48, No. 2, pages 121 to 130 *
R. Felleisen et al., "Analysis of a Cytovillin-Related Antigen from Echinococcus Multilocularis and E.Granulosus", Experientia, 1994, Vol. 50, page 76 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101949926A (zh) * 2010-08-04 2011-01-19 浙江大学 人体包虫病胶体金免疫层析试尿液快速诊断试纸卡
CN107884578A (zh) * 2017-11-01 2018-04-06 杭州微瑞科技有限公司 用于快速定量检测血清中包虫病抗体检测卡
CN109239211A (zh) * 2018-09-11 2019-01-18 江苏省血吸虫病防治研究所 用于鉴别人体感染包虫的血清标志物及检测试剂盒

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