| Project/Area Number |
10671490
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Urology
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| Research Institution | NARA MEDICAL UNIVERSITY |
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Principal Investigator |
HIRAO Yoshihiko (1999) Dep.Urol.Nara Med.Univ., Prof., 医学部, 教授 (00133207)
仲川 嘉紀 (1998) 奈良県立医科大学, 医学部, 助手 (60281276)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Kazuhiro 2nd Dept.Pathol.Aich Med.Univ., Assist.Prof., 医学部, 講師 (60109759)
UEMURA Hirotsugu Dep.Urol.Nara Med.Univ., Assist Prof., 医学部, 講師 (90213397)
平尾 佳彦 奈良県立医科大学, 医学部, 教授 (00133207)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Renal cell carcinoma / MN / CA9 / antibody |
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| Research Abstract |
MN/CA9 expression renal cell carcinoma cell line : This plasmid ligated MN/CA9 cDNA and BCMGSNeo vector introduced into RenCa cell, i.e.mouse rental cell carcinoma(RCC) cell line. Transfected RenCa cell clones were selected by growth in the presence of antibiotics G418 and expression of MN/CA9 was examined with mixed hemadsorption method.
MN/CA9 expression mouse RCC model. MN/CA9 expression RenCa cells were injected BALB/c mouse subcutaneously. The MN/CA9 expression of grown tumors was stained immunohistochemically with G250 antibody (G250 is already established MN/CA9 antibody).
MN/CA9 recombinant antigen : Specific regions of MN/CA9 DNA dissimilar to other antigen by amino acid homology comparisons was amplified by Polymerase chain reaction. The specific region joined expression vector PET32c and transformed E.coli. The protein produced in E.coli was analyzed by SDS-PAGE and western blotting and the examination displayed MN/CAIX expression of the protein.
Polyclonal antibody :. For production of polyclonal antibody, rabbit was immunized with synthesized MN/CAIX antigen emulsified with incomplete Freund adjuvant. The binding of antiserum with MN/CAIX antigen was measured in ELISA. Now antibody is isolated from the antiserum using affinity chromatography.
Monoclonal antibody : Hybridoma for monoclonal antibody was made using SP2/0 cells and spleen cells from mice immunized with cell lysate of RenCa cell expressed MN/CAIX. Specificity of monoclonal antibody was analyzed by using immunohistochemistry and confirmed to be similar to G250 antibody.
Single chain antibody : DNA of monoclonal antibody was cut in hybridoma gene and recombinant DNA molecule transferred to E.coli. E.coli made protein of single chain antibody. Specific single chain antibody is isolated from the protein and the affinity is analyzed. Furthermore, examination of single chain antibody for clinical use is now in progress.
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