| Project/Area Number |
12470239
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | Osaka University |
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Principal Investigator |
MIYAGAWA Shuji Osaka university graduate School of Medicine Associate Professor, 医学系研究科, 助教授 (90273648)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Hiroshi The Animal Engineering Research Institute Research maneger, 研究員
OKABE Masaru Genome Information research center Osaka Universigy Professor, 遺伝情報実験施設, 教授 (30089875)
SHIRAKURA Ryota Osaka university graduate School of Medicine Professor, 医学系研究科, 教授 (00116047)
FUKUTA Daisuke Nipro Corporation Research & Development Laboratory Researcher, 研究員
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2002: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Keywords | DAF / C1-INH / Complement regulatory protein / pig endothelial cell / CHO cell / HSV-gC1 / トランスジェニックマウス / Factor I / ブタ血管内皮細胞 / 細胞傷害性試験 |
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| Research Abstract |
We constructed a cell surface-bound form of a fluid phase CRP. Factor I-Pl
The cell membrane-bound form of factor I (fl-Pl) was constructed. An SEC line with a low expression of fl-Pl showed a weak inhibition of cell lysis in human serum, whereas a CHO cell transfectant with a high expression of fl-Pl showed over a 60% inhibition of cell lysis.
C1-INH
A surface-bound form of human C1-INH (C1-INH-Pl) was constructed Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-Pl showed a medium level of expression of these molecules. C1-INH-Pl block human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively.
The codon-optimization DAF
The DAF gene was synthesized in order to adapt its codons for those which are more frequent in mammals, especially pigs.
A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF. Consistent with the in vitro data, the expression of DAF in the mice which carry sDAF was higher than wild-type DAF in many organs.
Assessment of the effect of various forms of C1-INH-Pl and DAF
cDNAs of various deletion mutants of the C1-INH-Pl were established. While all deletion mutants of C1-INH-Pl except the delta-1-99AA were not expressed on the cell surface. Consequently, both the delta1-99AA C1-INH-Pl and delta-SCR1 DAF molecules are quite effective in downregulating the xenogeneic cell lysis, but accomplished this in different manners.
HSV-gC1
The complement regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule was investigated. The CHO cell transfectants showed a significant inhibition in cell lysis by the sera except for those that contained the preexisting anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked inhibition of cell lysis in all cases.
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