荧光素酶

National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20878,

Proteins engineered to generate fluorescent or bioluminescent signals are commonly used for the non-invasive study of a variety of biological processes and diseases in animals and humans (1-3). Usually the enhanced green fluorescent proteins, the variant red or yellow fluorescent proteins, or the bioluminescent luciferase proteins are used as reporter molecules because they produce a photo signal upon stimulation that can be tracked for the study of a dynamic biological system or phenomenon under and conditions (4-6). The luciferase family is composed of several different enzymes that catalyze light-producing, oxygen-dependent molecular reactions and have been detected across a diverse group of organisms including bacteria, fungi, insects, and marine animals (7). Among the luciferases, the firefly () luciferase (FLuc) has been extensively studied; it requires ATP and Mg as co-factors to function and oxidizes D-luciferin, its substrate, to release energy in the form of photons (8). The sea pansy () is another luciferase (RLuc) that has been extensively investigated; RLuc uses coelenterazine as a substrate and does not require ATP to produce light. However, RLuc has a lower quantum yield compared with FLuc (6% 88%) and also shows a low enzymatic efficiency (9). With the use of commercially available molecular kits, the humanized forms of FLuc and RLuc (designated as hFLuc and hRLuc, respectively) were combined as dual reporters to image cultured cells because both enzymes used different substrates (10, 11). However, these enzymes have very specific requirements to catalyze reactions under conditions to generate sufficient signal/background ratios for imaging. Tannous et al. decided to explore the possibility of finding a superior luciferase that was more efficient and had a higher quantum yield compared with the FLuc and RLuc enzymes (12). , a marine copepod, naturally secretes a luciferase (GLuc) that was cloned, expressed in , and used as a detection agent in a DNA hybridization assay (13). Tannous et al. cloned the humanized version of the GLuc enzyme (hGLuc) into a herpes simplex virus-1 (HSV-1) amplicon under a cytomegalovirus (CMV) immediate early (IE) promoter and evaluated the bioluminescence emitted from DNA-transfected and vector-infected mammalian cells (12). The luciferase was also evaluated for imaging cultured cells after subcutaneous implantation of GLuc-transfected cells into nude mice (12). In another study, GLuc was evaluated for imaging after it was cloned into a lentivirus (LV) vector and transduced into human glioma cells that were implanted into nude mice (14).

经过工程改造以产生荧光或生物发光信号的蛋白质通常用于对动物和人类的各种生物过程及疾病进行非侵入性研究(1 - 3)。通常，增强型绿色荧光蛋白、红色或黄色荧光蛋白变体或生物发光的荧光素酶蛋白被用作报告分子，因为它们在受到刺激时会产生光信号，可用于追踪研究动态生物系统或现象在特定条件下的情况(4 - 6)。荧光素酶家族由几种不同的酶组成，这些酶催化产生光的、依赖氧气的分子反应，并且已在包括细菌、真菌、昆虫和海洋动物在内的多种生物中被检测到(7)。在这些荧光素酶中，萤火虫（Photinus pyralis）荧光素酶（FLuc）已得到广泛研究；它需要ATP和Mg作为辅助因子来发挥作用，并氧化其底物D - 荧光素，以光子的形式释放能量(8)。海肾（Renilla reniformis）荧光素酶（RLuc）是另一种已被广泛研究的荧光素酶；RLuc使用腔肠素作为底物，产生光不需要ATP。然而，与FLuc相比，RLuc的量子产率较低（6%对88%），并且酶效率也较低(9)。利用市售的分子试剂盒，将人源化形式的FLuc和RLuc（分别命名为hFLuc和hRLuc）组合作为双报告分子来对培养细胞进行成像，因为这两种酶使用不同的底物(10, 11)。然而，这些酶在特定条件下催化反应以产生足够的信号/背景比用于成像时具有非常特殊的要求。Tannous等人决定探索寻找一种比FLuc和RLuc酶更高效且量子产率更高的优质荧光素酶的可能性(12)。海萤（Gaussia princeps）是一种海洋桡足类动物，它天然分泌一种荧光素酶（GLuc），该酶被克隆、在细胞中表达，并在DNA杂交检测中用作检测剂(13)。Tannous等人将GLuc酶的人源化版本（hGLuc）克隆到巨细胞病毒（CMV）立即早期（IE）启动子控制下的单纯疱疹病毒1型（HSV - 1）扩增子中，并评估了DNA转染和载体感染的哺乳动物细胞发出的生物发光(12)。在将GLuc转染的细胞皮下植入裸鼠后，还对该荧光素酶用于成像培养细胞进行了评估(12)。在另一项研究中，在将GLuc克隆到慢病毒（LV）载体中并转导到植入裸鼠的人胶质瘤细胞后，对其成像进行了评估(14)。