Recombinant expression and functional analysis of the protein encoded by Mce 1A region of Mycobacterium leorae
| Project/Area Number |
15591190
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Dermatology
|
|---|
| Research Institution | Kitasato 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)
|
|---|
| Keywords | Mycobacterium 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)
Research Products
(8 results)
