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Recombinant expression and functional analysis of the protein encoded by Mce 1A region of Mycobacterium leorae

Research Project

Project/Area Number 15591190
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Dermatology
Research InstitutionKitasato University

Principal Investigator

FUJIMURA Takao  Kitasato University, Sch. of Med., Dermatology, Assist. Prof. (50209087)

Co-Investigator(Kenkyū-buntansha) MASUZAWA Mikio  Kitasato University, School of Med., Dermatology, Associate Prof. (30129267)
YOGI Yasuko  National Institute of Infectious Diseases, Leprosy Research Center, Chief (10210600)
宮田 聡子  北里大学, 医学部, 講師 (30260855)
Project Period (FY) 2003 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2006: ¥100,000 (Direct Cost: ¥100,000)
Fiscal Year 2005: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,600,000 (Direct Cost: ¥1,600,000)
KeywordsMycobacterium leprae / mce1A gene / AIDA / Mammalian cell entry / Virulence factor / AIDA
Research Abstract

Mce1A protein is coded in mce1A locus in M. leprae. Mce1A protein is known to be associated with ability of M. tuberculosis to enter epithelial cells and to survive and multiply within macrophages. The M. leprae genome has a region which is highly homologous with mce1A region of M. tuberculosis. Studies using recombinant proteins indicate this region is associated with M. leprae epithelial cell entry. This study is aimed at identifying the active sequence of mce1A associated with M. leprae epithelial cell entry.
Recombinant proteins where the N terminus and C terminus of mce1A region of M. leprae (1326bp and 442aa) are truncated were created in E. coli. FITC labeled polystyrene beads and latex beads were coated with each truncated protein (r-lep37KDa and r-lep27KDa). The entry activity into HeLa cells was observed using a fluorometer and an electron microscope. The entry activity was preserved even when 316bp (105aa) was truncated from N terminus and 921 bp (307aa) was truncated from C terminus. The 316-921bp region was divided into three sub-regions: 316-531bp, 532-753bp and 754-921bp. Each subdivided region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of recombinant E. coli into monolayer-cultured HeLa and BEAS-2B cells were observed using an electron microscope. Recombinant E. coli were treated with anti-r-lep27KDa antibody and added to the monolayer-cultured BEAS-2B cells. The effectiveness of the antibody for prevention of cell entry was studied by colony count. Cell entry was observed only for E-coli harboring region of 316-531bp. This entry activity is suppressed by the antibody. A portion of the peptide in 316-531bp region (388-453bp) was synthesized and conjugated to colloidal gold particles. Observation by an electron microscope revealed that the synthesized peptide entered the BEAS-2B cells. These findings indicate that active sequence within 316-531bp region is 388-453bp.

Report

(5 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (8 results)

All 2007 2006

All Journal Article (6 results) Patent(Industrial Property Rights) (2 results)

  • [Journal Article] Recombinant Mycobacterium leprae protein associated with entry into mammalian cell of respiratory and skin components2007

    • Author(s)
      Naoya Sato, Takao Fujimura, et. al.
    • Journal Title

      Journal of Dermatological Science 46

      Pages: 101-110

    • NAID

      10024116033

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components.2007

    • Author(s)
      Naoya Sato, Takao Fujimura, Mikio Masuzawa, Yasuko Yogi, Masanori Matsuoka, Maha Kanoh, Lee W. Riley, Kensei Katsuoka
    • Journal Title

      Journal of Dermatological Science(Epub) 46(2)

      Pages: 101-10

    • NAID

      10024116033

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components2007

    • Author(s)
      Naoya Sato, Takao Fujimura, et al.
    • Journal Title

      Journal of Dermatological Science online

      Pages: 10-10

    • NAID

      10024116033

    • Related Report
      2006 Annual Research Report
  • [Journal Article] らい菌mce1A遺伝子にコードされる蛋白はヒト上皮細胞への侵入に関与する重要な病原因子である2006

    • Author(s)
      佐藤 直哉, 藤村 響男, ら
    • Journal Title

      北里医学 36

      Pages: 65-73

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Mycobacterium leprae protein encoded by the mcelA gene is an important virulence factor associated with mammalian epithelial cell entry.2006

    • Author(s)
      Naoya Sato, Takao Fujimura, Yasuko Yogi, Maho Kanoh, Mikio Masuzawa, Kensei Katsuoka
    • Journal Title

      Kitasato Medicine 36

      Pages: 65-73

    • NAID

      110006405960

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] らい菌mce1A遺伝子にコードされる蛋白はヒト上皮細胞への侵入に関与する重要な病原因子である2006

    • Author(s)
      佐藤直哉, 藤村響男 et al.
    • Journal Title

      北里医学 36

      Pages: 65-73

    • Related Report
      2006 Annual Research Report
  • [Patent(Industrial Property Rights)] 細胞内に侵入する能力を有するペプチド及び核酸, 物質を細胞内に侵入させるための物質移送用剤及び物質移送方法, 並びに, らい菌による感染症を予防又は治療するためのワクチン, 抗体及び医薬2006

    • Inventor(s)
      藤村 響男
    • Industrial Property Rights Holder
      北里学園
    • Industrial Property Number
      2006-347090
    • Filing Date
      2006-12-25
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Patent(Industrial Property Rights)] 細胞内に侵入する能力を有するペプチド及び核酸、物質を細胞内に侵入させるための物質移送用剤及び物質移送方法、並びに、らい菌による感染症を予防又は治療するためのワクチン、抗体及び医薬2006

    • Inventor(s)
      藤村, 響男
    • Industrial Property Rights Holder
      北里学園
    • Industrial Property Number
      2006-347090
    • Filing Date
      2006-12-25
    • Related Report
      2006 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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