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Production and evaluation of recombinant allergens with carbohydrate epitopes expressed in insect cells

Research Project

Project/Area Number 12556060
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Applied molecular and cellular biology
Research InstitutionNAGOYA UNIVERSITY

Principal Investigator

MATSUDA Tsukasa  Nagoya University, Graduate School of Bioagricultural Sciences, Professor, 大学院・生命農学研究科, 教授 (20144131)

Co-Investigator(Kenkyū-buntansha) AOKI Naohito  Nagoya University, Graduate School of Bioagricultural Sciences, Assistant Professor, 大学院・生命農学研究科, 助手 (40242846)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 2001: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2000: ¥3,300,000 (Direct Cost: ¥3,300,000)
KeywordsAllergy / Carbohydrate antigen / Insect cells / Plant allergens
Research Abstract

Several glycoproteins with asparagine-linked sugar chains were expressed in insect cells, and the glycoproteins with fucose-containing sugar chains were produced. First, the cDNA encoding each protein was inserted into a transfer vector to introduce into baculovirus genome DNA. The viral genome DNA and the transfer vector containing the CDNA were introduced into a insect cell line, BTI TN 5B1-4. The virus particles were recovered from the culture supernatant, and its betagalactosidase activity was measured. The galactosidase-positive recombinant virus was selected and cloned. Each recombinant virus was cultured in a larger scale, and recombinant glycoproteins expressed in the insect cells were purified. To determine whether sugar chains with the carbohydrate epitopes were added to the recombinant proteins, the purified glycoproteins were subjected to immunoblotting and ELISA using the antiserum specific for the carbohydrate epitope. Both of the blot and ELISA demonstrated that all the recombinant glycoproteins tested were clearly positive to the carbohydrate epitope-specific antibody, though the reactivity varied from one protein and another. Furthermore, the presence of the carbohydrate epitope was confirmed from the observation that the antigenic reactivity of each recombinant glycoprotein was lost by the periodate treatment. Further detailed analyzes to characterize the carbohydrate structure are in progress including lectin-binding assay, glycosidase treatments, chemical analysis, and immunochemical analyzes using patient's antibodies. The present research demonstrated that recombinant glycoprotein allergens with carbohydrate epitopes can be produced in the baculovirus/insect cell expression system, and suggests that the recombinant proteins show allergenic reactivity comparable to natural allergens. Thus, baculovirus/insect cell expression system would be a useful tool for theproduction of glycoprotein allergens for research and diagnosis purposes.

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (30 results)

All Other

All Publications (30 results)

  • [Publications] Kato, Y. et al.: "Ovomucoid rendered insolble by heating with wheat gluten but not with milk casein"Biosci. Biotechnol. Biochem. 64(1). 64. 198-201 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sato, C. et al.: "Frequent occurrence of pre-existing α2-8-linked dosialic and oligosialic acids with chain lengths up to 7 Sia residues in mammalian brain glycoproteins"J. Biol. Chem.. 275. 15422-15431 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Oubihi, M. et al.: "An ELISA-based assay for detergent-solubilized cellular B1, 4-galactosyltransferase activity. Utilization of a polyacrylamide derivative with GlcNAcB side chains as a solid phase acceptor"Biosci. Biotechnol. Biochem.. 64. 785-792 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Oubihi, M. et al.: "Spontaneous galactosylation of agalactoglycoproteins in colostrum"FEBS Letters. 473. 165-168 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakata, D. et al.: "Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which has not the deaminoneuraminic acid (KDN) 9-phosphate synthase activity"Biochem. Biophys. Res. Commun.. 273. 642-648 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, T. et al.: "Release of allergenic proteins from rice grains induced by high hydrostatic pressure"J. Agric. Food Chem.. 48. 3124-3129 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Izumi, H. et al.: "Decrease in rice allergenic proteins of polished rice grains by incubating with a miso solution"Biosci, Biotechnol. Biochem.. 64. 2248-2251 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Usui, Y. et al.: "A 33kDa allergen from rice (Oryza sativa L.Japonica) : cDNA cloning, expression and identification as a novel glyoxalase I"J. Biol. Chem.. 276. 11376-11381 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, Y. et al.: "Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour : dependence on wheat variety and disulfide Bridges"J. Agric. Food Chem.. 49. 3661-3665 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakata, D. et al.: "Molecular cloning of a unique CMP-sialic acid synthetase that effectively utilizes both KDN and Neu5Ac as substrate"Glycobiology. 11. 685-692 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, Y., Watanabe, H., and Matsuda, T: "Ovomucoid rendered insolble by heating with wheat gluten but not with milk casein"Biosci. Biotechnol. Biochem. 64. 198-201 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi, K: "Frequent occurrence of pre-existing a2-8-linked disialic and oligosialic acids with chain lengths up to 7 Sia residues in mammalian brain glycoproteins"J. Biol. Chem. 275. 15422-15431 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Oubihi, M., Oshima, K., Aoki, N., Kobayashi, K., Kitajima, K. and Matsuda, T: "An ELISA-based assay for detergent-solubilizedcellular bl ,4-galactosyltransferase activity. Utilization of a polyacrylamide derivative with GlcNAc-b side chains as a solid phase acceptor substrate"Biosci. Biotechnol. Biochem. 64. 785-792 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Oubihi, M., Kitajima, K., Aoki, N. and Matsuda, T: "Spontaneous galactosylation of agalactoglycoprotein in colostrum"FEBS Letters. 473. 165-168 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakata, D., Close, B.E., Colley, K.J., Matsuda, T., and Kitajima, K: "Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity"Biochem. Biophys. Res. Commun. 273. 642-648 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, T., Katayama, A., Matsubara, S., Oumi, Y. and Matsuda, T: "Release of allergenic proteins from rice grains induced by high hydrostatic pressure"J. Agric. Food Chem. 48. 3124-3129 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Izumi, H., Kondo, S., Kasho, H., Matsuda, T. and Nakamura, R: "Decrease in rice allergenic proteins of polished rice grains by incubating with a miso solution"Biosci. Biotechnol. Biochem. 64(10). 2248-2251 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Usui, Y., Nakase, M., Hotta, H., Urisu, A., Aoki, N., Kitajima, K., and Matsuda, T: "A 33 kDa allergen from rice (Oryza saliva L. Japonica) : cDNA cloning, expression and identification as a novel glyoxalase I"J. Biol. Chem. 276. 11376-11381 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, Y., Oozawa, E., and Matsuda, T: "Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour : dependence on wheat variety and intermolecular disulfide bridges"J. Agric. Food Chem. 49. 3661-3665 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Nakata, D., Munster, A.-K., Gerady-Schahn, R., Aoki, N., Matsuda, T., and Kitajima, K: "Molecular cloning of a unique CMP-sialic acid syntase that effectively utilizes both deaminoneuraminic acid (KDN) and N-acetylneuraminic acid (Neu5Ac) as substrates"Glycobiology. 11. 685-692 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Kato, Y. et al.: "Ovomucoid rendered insoluble by heating with wheat gluten but not with milk casein"Biosci. Biotechnol. Biochem. 64(1). 64. 198-201 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Sato, C. et al.: "Frequent occurrence of pre-existing α 2-8-linked disialic and oligosialic acids with chain lengths up to 7 Sia residues in mammalian brain glycoproteins"J. Biol. Chem.. 275. 15422-15431 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Oubihi, M. et al.: "An ELISA-based assay for detergent-solubilized cellular β 1,4-galactosyltransferase activity Utilization of a polyacrylamide derivative with GlcNAc β side chains as a solid phase acceptor"Biosci. Biotechnol. Biochem.. 64. 785-792 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Oubihi, M. et al.: "Spontaneous galactosylation of agalactoglycoproteins in colostrum"FEBS Letters. 473. 165-168 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nakata, D. et al.: "Molecular cloning and expression of the mouse N-acetylneuraminic acid 9-phosphate synthase which does not have deaminoneuraminic acid (KDN) 9-phosphate synthase activity"Biochem. Biophys. Res. Commun.. 273. 642-648 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kato, T. et al.: "Release of allergenic proteins from rice grains induced by high hydrostatic pressure"J. Agric. Food chem.. 48. 3124-3129 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Izumi, H. et al.: "Decrease in rice allergenic proteins of polished rice grains by incubating with a miso solution"Biosci. Biotechnol. Biochem.. 64. 2248-2251 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] Usui, Y. et. al.: "A 33kDa allergen from rice (Oryza sativa L. Japonica) : cDNA cloning, expression and identification as a novel glyoxalase I"J. Biol. Chem.. 276. 11376-11381 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Kato, Y. et al.: "Decrease in antigenic and allergenic potentials of ovomucoid by heating in the presence of wheat flour: dependence on wheat variety and disuldide briages"J. Agric. Food Chem.. 49. 3661-3665 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nakata, D. et al.: "Molecular cloning of a unique CMP-sialic acid syntase that effectively utilizes both KDN and Neu5Ac as substrates"Glycobiology. 11. 685-692 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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