Exhaustive analyses of mechanism of plant cell death using fluorobody

Research Project

Project/Area Number	26670046
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Natural medicines
Research Institution	Kyushu University
Principal Investigator	Morimoto Satoshi 九州大学, 薬学研究科(研究院), 教授 (60191045)
Co-Investigator(Kenkyū-buntansha)	TANAKA Hiroyuki 九州大学, 薬学研究院, 准教授 (30253470) SAKAMOTO Seiichi 九州大学, 薬学研究院, 助教 (50610177)
Project Period (FY)	2014-04-01 – 2016-03-31
Project Status	Completed (Fiscal Year 2015)
Budget Amount *help	¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000) Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywords	細胞死 / モノクローナル抗体 / ｓｃFV / fluorobody / cytochrome c / cyclophilin D / VDAC / アポトーシス / scFV
Outline of Final Research Achievements	I cloned and expressed genes encoding three factors (cyclophilin D, CycD; voltage-dependent anion channel, VDAC; cytochrome c, Cytc), which are participated with plant cell death. To prepare the monoclonal antibodies against these factors, I injected these recombinant proteins. As a result, I could obtain the monoclonal antibodies, which highly specifically bind to each antigen. Next, I constructed the gene encoding scFV against these factors, and succeeded in the expression of these genes in E. coli. CycD gene, which was cloned in this study, was found to be not involved in cell death, because the recombinant CycD did not bind to cyclosporine A. Therefore, I attempt to clone the genes encoding the CycD actually mediating cell death in plant and to prepare monoclonal antibody against this CycD. Now I attempt to construct fluorobody genes by connecting scFV genes with green-fluorescent-protein gene, and develop genetically engineered plant by introducing the fluorobody.