GFP，螢光蛋白的發現為研究者提供了有利的工具，常用於研究小分子在生物體內的功能性或是位置，除了上述的應用外還可用於研究蛋白質的交互作用，例如:螢光共振能量轉移( FRET )，目前已有許多的藍色螢光蛋白被開發但仍存在low quantum yields與photostability的問題，我們從Vibrio vulnificus找到的藍色螢光蛋白dBP6，經由胺基酸序列比對發現同源於SDR家族，其發光機制是藉由結合上NADPH放大內生性螢光，利用E. coil表現大量dBP6蛋白並測其螢光光譜，得知該蛋白的最大激發波長為335 nm 而放射波長為450 nm，此蛋白的放射波長很接近GFP的激發波長(~488 nm) 所以我們想要在FRET應用以dBP6作為GFP的donor，為此我們將dBP6與EGFP以數個胺基酸相連並在之中設計caspase-3辨認切位為設計的質體命名為mBcg，在H1299中表現mBcg經UV激發可以偵測到GFP的訊號，表示有FRET的現象發生，而在活化caspase-3之後，可以看到綠色螢光逐漸消失而同時地藍色螢光漸漸出現；另一方面，我們利用已知有交互作用的蛋白ribosomal phosphoprotein p0與GCIP來證實以dBP6與EGFP作FRET配對的可行性，在UV激發下可以觀察到在細胞質的部分有綠色螢光；從GFP衍伸而來的螢光蛋白其發光機制需要氧的參與，而dBP6的輔因子是NADPH，結果不管在細胞或是細菌中dBP6可以在厭氧環境中產生螢光而其他的螢光蛋白則無法，所以dBP6在生物醫學上的應用是具有發展潛力。

Fluorescence proteins (FPs), such as green fluorescent proteins (GFPs) are used in a variety of applications to study the organization and function of living systems. FPs encoded in frame with proteins of interest made it possible to observe their localization, movement, turnover, and aging. Moreover, complex functional studies can be performed using FPs such as visualizing protein-protein interactions in living cells by FRET (Fluorescence Resonance Energy Transfer) detection technique. Although a few blue fluorescent proteins derived from GFP have been developed, they still have low quantum yields and poor photostability. In addition, all members of GFP family have a major drawback-their strict requirement for molecular oxygen as a cofactor for the synthesis of their respective chromophores. The goal of this study is to evaluate whether our developed BFP (dBP6) could be used for in vivo labeling and detection in both the presence and absence of oxygen. dBP6 has the excitation/emission peaks at 352/456 nm and it is derived from BFPvv, a FP isolated from bacterium and fluoresces through augmenting the intrinsic fluorescence of NADPH bound to it chromophore. To achieve our goal, plasmid mBcg that contains dBP6 and EGFP with the caspase-3 cleavage sequence, DEVD, within the linker between fluorescent proteins, was constructed and transfected into mammalian cell H1299, and its fluorescence was examined. The results showed that H1299 containing mBcg exhibited green fluorescence when irradiated by UV source. After activation of caspase-3, cleavage of mBcg led to a decrease in green fluorescence of EGFP and a simultaneous increase in blue fluorescence of dBP6 was observed. On the other hand, our previous data revealed that GCIP could interact with ribosomal phosphoprotein p0. We constructed a plasmid dBP6-GCIP and a plasmid EGFP-P0, and cotransfected the plasmids dBP6-GCIP and EGFP-P0 into H1299. The results showed that transfected cells exhibited green fluorescence in cytoplasm when irradiated by UV source. In conclusion, through construction of a variety of plasmids and the examination of their fluorescence under various conditions, dBP6 is founded to be applicable in mammalian cells in vivo labeling under aerobic and anaerobic conditions.

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