Development of New concept for the evaluation of protein-or gene-function

本研究的目的是为蛋白质或基因功能的评价提供新的概念。在这方面，研究了以下四类：1：蛋白质固定化生物传感器，2：使用2D-SPR生物芯片的细胞内信号检测系统，3：使用质谱的细胞内信号评估系统，4：在蛋白质固定化生物传感器中，用镍-NTA络合物将甲状腺激素受体（TR）和二恶英受体（AhR）通过寡聚组氨酸标记固定在金电极上。这些传感器响应于它们的配体，包括甲状腺素，萘酚和苯并芘。在2D-SPR系统中，蛋白激酶A、蛋白激酶C和Src的特异性底物通过马来酰亚胺基团固定在金表面N-端半胱氨酸残基的巯基上。使用抗磷酸酪氨酸抗体用2D-SPR成像仪对获得的芯片成像。利用该系统可以成功地监测蛋白激酶的磷酸化反应。该系统可用于细胞内信号的分析。在质谱系统中，可以用PC-12细胞评价各种药物作用后PKA活性的变化。并与报告基因检测结果进行了比较。这两个结果都具有很好的可行性。这意味着他的方法可以用作细胞功能的评价系统。PKC和Src活性也能够使用该系统进行监测。在利用微孔进行表面等离子体共振传感器的研究中，从理论上进行了探讨，建立了微孔表面等离子体共振现象的理论。

Objective of the research is to develop new concepts for the evaluation of protein-or gene-functions. In this term, following four categories were investigated ; 1 : protein immobilized biosensor, 2 : intracellular signal-detecting system using 2D-SPR biochip, 3 : intracellular signal-evaluating system using mass spectrometry, 4 : Theoretical consideration of SPR on micro-corn, In the protein-immobilized biosensor, thyroid hormon receptor (TR) and dioxin receptor (AhR) were immobilized on a gold electrode through oligohistidine-tag using nickel-NTA complex. These sensors responded to their ligands including thyroxine, naphthoflavon and benzopyrene. The detection sensitivities of these sensors were comparable to those of Rl methods, In the 2D-SPR system, specific substrate of protein kinase A, protein kinase C and Src were immobilized on the gold surface at the thiol group in the N-terminus-cysteine residue using maleimide group on the surface. obtained chip was imaged with 2D-SPR imager using anti-phosphotyrosin antibody. Phosphorylation reaction with protein kinase could be successfully monitored using this system. This system can be a good method for the profiling of intracellular signals. In the mass spectrometric system, the change of PKA activity could be evaluated using PC-12 cell after the treatment of various drugs. This results were compared with those of reporter gene assay. Both results had good feasibility. This means that his method can be used as the evaluation system of cellular function. PKC and Src Activities were also able to be monitored using this system. In the investigation of the SPR sensor using micro-corn, theoretical consideration was investigated and the theory of SPR phenomena on such microarea could be established.