Luciferase refers to a group of bioluminescence producing enzymes that enable researchers to study gene expression and regulation.

蟲螢光素酶是指，一群產生生物冷光，使研究人員們能研究基因表現及調節的酵素。

圖1. 發光的螢火蟲，在蟲螢光素酶的酵素協助下，照亮森林。

Glowing fireflies illuminate a forest with the help of luciferase enzymes.

Luciferase is a group of enzymes that oxidize a substrate known as luciferin to produce light.  For bioluminescence, the firefly luciferase enzyme catalyzes the oxidation of D-luciferin in the presence of oxygen (O₂), adenosine triphosphate (ATP), and magnesium ions (Mg²⁺).

蟲螢光素酶是一群，氧化一種被通稱為蟲螢光素之酶作用物(因酶或酵素的作用，引起化學反應的物質)，以產生光的酵素。就生物冷光而言，螢火蟲的蟲螢光素酶酵素，在氧(O₂)、三磷酸腺苷(ATP)及鎂離子(Mg²⁺)存在的情況下，催化D-蟲螢光素的氧化。

This catalytic reaction converts the excited oxyluciferin molecule to its ground state, emitting visible light. Bioluminescence observed in various organisms may utilize different chemical processes. However, they all share a common feature: the production of light through enzyme-catalyzed oxidation reactions.

此催化反應將被激發的氧蟲螢光素分子，轉變到發出可見光的基態。於各種生物體中，被觀察到的生物冷光，可能利用不同的化學變化過程。不過，它們全有種共同特性：也就是，光的產生是透過酵素催化的氧化反應。

Although bioluminescence has been observed for many centuries, its application in biomedicine is relatively recent. Scientists traced back the first reference of bioluminescence to the Greek philosopher Aristotle in 384-322 BCE.

雖然，生物冷光已經被觀察，達幾個世紀。不過，其在生物醫學方面的應用，相對上是近代的。科學家們將生物冷光的首次提及，追溯到公元前(Before the Christian Era)384-322年的希臘哲學家亞里斯多德。

 Almost three centuries later, Gaius Plinius Secundus, a Roman author, conducted an in-depth study on bioluminescence. He discovered many animals that had illuminating capacity such as purple jellyfish, mollusks, fireflies, and glowworms. 

將近三個世紀後，羅馬作家Gaius Plinius Secundus，針對生物冷光進行了深入研究。他發現了，許多具有發光能力的動物，諸如紫色水母、軟體動物、螢火蟲及發光蟲等。

French pharmacologist Raphael Dubois observed that a specific constituent of the click beetle was essential for bioluminescent reactions. He extracted two components from beetle abdomens, naming the substrate luciferine and the enzyme responsible for the reaction luciferase.

法國藥理學家Raphael Dubois觀察到，磕頭甲蟲的一種特定成分，對於生物冷光反應是不可或缺的。他從甲蟲腹部提取了兩種成分，將此酶作用物命名為蟲螢光素，而此酵素導致上述蟲螢光素酶反應。

Over many years, scientists across the world conducted multiple experiments and established that luciferase enzymes and substrates are species-specific. For example, the Gaussia and Renilla luciferases use coelenterazine as their substrate to emit blue to cyan light, while Cypridina uses its own genus-specific luciferin to generate blue light.

多年來，世界各地的科學家們，進行了多次實驗且證實，蟲螢光素酶及酶作用物是物種-特有的。譬如，Gaussia及Renilla蟲螢光素酶，使用腔腸素作為它們發出藍色到青色光的酶作用物，而Cypridina使用本身之屬-特有的蟲螢光素，來產生藍光。

In 1985, scientists made a milestone scientific breakthrough by cloning the firefly luciferase (FLuc) in Escherichia coli, enabling an unlimited source for this enzyme.  Different types of luciferases are present in aquatic and terrestrial species including insects, bacteria, and fungi. Scientists estimated that around 75% of sea animals are bioluminescent.

於1985年，科學家們藉由，在大腸桿菌中，無性生殖了螢火蟲的蟲螢光素酶(FLuc)，獲得了科學突破性里程碑，使其能作為此種酵素的一種無限來源。不同類型的蟲螢光素酶存在於，包括昆蟲、細菌及真菌等，水生與陸生物種中。科學家們估計，大約75%的海洋動物是發生物冷光的。

In nature, a luciferin–luciferase system is present in the firefly (Photinus pyralis), bacteria (Vibrio harveyi), sea pansy (Renilla reniformis), jellyfish (Aequorea victori), ostracod (Cypridina noctiluca), dinoflagellates (Gonycaulax), and copepod (Gaussia princeps).

在自然界中，有種蟲螢光素與蟲螢光素酶物的物系存在於，螢火蟲(Photinuspyralis：東方螢火蟲)、細菌(Vibrio harveyi：哈維氏弧菌)、海堇(Renilla reniformis)、水母(Aequorea victori：維多利亞多管發光水母)、介形類(Cypridina noctiluca)、甲藻(Gonycaulax：膝溝藻)及橈足類(Gaussia princeps：高斯王子)中。

Not all bioluminescent marine organism systems can be used for animal imaging because the majority of them generate blue light that does not penetrate tissue appropriately for detection. Furthermore, certain small molecule substrates of marine organisms are unstable and easily oxidized without luciferase. 

並非所有發生物冷光的海洋生物物系，能被使用供作動物造影之用。因為，大多數它們產生，無法適當穿透組織，來供作探知之用的藍光。此外，海洋生物的某些小分子酶作用物不穩定，且在沒有蟲螢光素酶的情況下，容易被氧化。

Table 1: Different types of luciferase and their applications

表1：不同類型的蟲螢光素酶及其應用(請參閱原文)

Among the different types of luciferases, scientists use FLuc widely for biological experiments because of its high quantum yield, elevated signal to noise ratio, and magnificent color. Furthermore, these luciferases trigger yellow-green to red light emission, depending on reaction conditions, which has a higher cellular or tissue penetrance. Luciferin is biologically stable as it is not oxidized in the absence of the enzyme. 

在不同類型的蟲螢光素酶中，FLuc因其高量子產率、提高的信號對噪音比及絢麗的色彩，而被科學家們廣泛使用供作生物實驗之用。此外，這些蟲螢光素酶觸發，發出取決於反應條件，而具有較高細胞或組織穿透力之黃-綠到紅光。蟲螢光素生物學上是穩定的，因為沒有蟲螢光素酶，它不會被氧化。

Biochemical engineers created several generations of FLuc that exhibit improved brightness and other properties. For example, Luc2 is a second-generation firefly luciferase with an optimized codon that facilitates its expression in mammalian systems.

生化工程師們創造了，幾代展現出經改善亮度及其他屬性的FLuc。譬如Luc2是具有，在哺乳動物物系中促進其表現，經優化密碼子之第二代螢火蟲的蟲螢光素酶。

The new firefly bioluminescence system Akaluciferase (AkaLuc)-AkaLumine produces greater signal strength than the standard Fluc-luciferin reaction.

該種新的螢火蟲生物冷光物系，Akaluciferase (AkaLuc)-AkaLumine產生，比標準Fluc與蟲螢光素之反應，更強的信號。

AkaLuc-AkaLumine (named after the Japanese word for the color red, aka) is a non-invasive and highly sensitive bioluminescence system that emits red light in the near-infrared (NIR) range for better tissue penetration. Besides FLuc, researchers also use NanoLuc luciferase (NLuc) and Renilla luciferase (RLuc) for biological experiments.

AkaLuc-AkaLumine(是以日語代表紅色的別名)是一種，在近紅外線(NIR)範圍內發出紅光，供作更佳穿透組織之用，非侵入性、高度敏感的生物冷光物系。除了FLuc之外，研究人員們也使用，NanoLuc蟲螢光素酶(NLuc)及Renilla蟲螢光素酶(RLuc)，供作生物實驗之用。

Each luciferase type has different advantages and disadvantages depending on the application. For instance, both FLuc and RLuc have short protein half-lives making them favorable transcriptional reporters. Even though FLuc is brighter than RLuc, the former is more vulnerable to enzyme inhibition.

取決於應用，每一種蟲螢光素酶具有不同的優點及缺點。例如，FLuc及RLuc 兩者皆具有短的蛋白質半衰期，使它們成為良好的轉錄信息物。即使，FLuc比RLuc更亮。不過，前者較容易受到酵素抑制。

Because FLuc and RLuc are not secreted, transfected cells must be lysed, followed by intracellular substrate introduction and luminescence measurement. Furthermore, both FLuc and RLuc are large, making them less favorable protein tags.

由於FLuc及RLuc非分泌型，因此轉染的細胞(經引進外源之DNA、RNA 或蛋白質的細胞)必須經裂解，然後進行引入細胞內的酶作用物及發光測量。此外，FLuc及RLuc兩者皆屬大的，使得它們是較不佳的蛋白質標記。

The secreted Gaussia luciferase (GLuc) is more stable. However, a relatively short luminescence half-life makes it unsuitable for many experimental settings. NLuc is an engineered luciferase derived from deep sea luminescent shrimp.

分泌型Gaussia蟲螢光素酶(GLuc)較穩定。不過，是一種相對上短的發光半衰期，使其不適合許多實驗環境。NLuc是一種，源自深海發光蝦，經工程改造的蟲螢光素酶。

This reporter gene has significantly attracted scientists for its greater stability, smaller size, and >150-fold increase in luminescence. Despite these benefits, they are not ideal for in vivo applications because its blue emission and their substrate (furimazine) has low solubility and bioavailability. 

該信息基因，由於其較高的穩定性、較小的尺寸及在發光方面，增加超過150倍。因此，一直極大地吸引科學家們。儘管此些優點，不過就活體內的應用而言，它們並不理想。因為，其發出的藍光及其酶作用物(呋喃嗪)，具有低的溶解度及生物可資使用性。

The major advantage of a bacterial bioluminescence system is that, since researchers have figured out the genetic pathways to synthesize all of the substrates required for the emission of light, no exogenous luciferin addition is needed.

由於，研究人員們已經瞭解，合成發光所需之所有酶作用物的遺傳途徑。因此，細菌生物發光物系的主要優點是，無需添加外源的蟲螢光素。

 However, its dependence on reduced riboflavin phosphate (FMNH₂), which is available at a limited quantity in mammalian cells, and low intensity blue light emission makes it less favorable for eukaryotic systems. In addition, fungi possess a bioluminescent system with the synthetic genes elucidated, but the intensity of this system is still weak.

不過，它依賴於哺乳動物細胞中，經還原之數量有限的核黃素磷酸鹽(FMNH₂)，及發出低強度的藍光，使其較不適合真核細胞物系。此外，真菌擁有一種，具有經闡明之合成基因的生物發光物系。不過，該物系的強度仍然很弱。

Luciferase reporter assays help determine whether a protein activates or suppresses transcription of the target gene. It also analyzes translation regulation by cis-elements of an mRNA present in the 5′-untranslated region (UTR) and 3′-UTR. 

蟲螢光素酶信息測定有助於，確定蛋白質是否活化或抑制標的基因的轉錄。它也藉由，存在於 5'-非轉譯區(UTR)及3'-UTR中，mRNA的順式元件(能與轉錄因子結合並影響附近基因表現的短DNA片段)，來分析轉譯調節。

圖2. 蟲螢光素信息測定，採用蟲螢光素酶與蟲螢光素的發光反應，作為標的基因啟動子活性及基因表現的一種信息讀出。

Luciferin reporter assays employ a luciferase-luciferin light emitting reaction as a readout for target gene promoter activity and gene expression.

Scientists use recombinant DNA technology to develop promoter-reporter constructs. They fuse the regulatory region of the target gene with a luciferase reporter gene. A second DNA construct encodes proteins hypothesized to influence transcription. Researchers transfect a cell culture system such as HEK 293T cells with both constructs.

科學家們使用重組的DNA技術，來開發啟動子與信息的構成物。他們使標的基因的調節區域與一種蟲螢光素酶信息基因融合。第二種DNA構成物編碼，被推測影響轉錄的蛋白質。研究人員們以這兩種構成物，轉染諸如HEK 293T細胞(衍生自人胚胎腎細胞的細胞)等，細胞培養物系。

Typically, scientists examine luciferase expression two to three days after cell transfection. They lyse the transfected cells and place the contents in a reaction tube. Researchers add a suitable substrate to the reaction tube, such as luciferin, which enables luciferase to catalyze a chemical reaction that produces light.

通常，在細胞轉染後，科學家們檢視蟲螢光素酶表現，兩到三天。他們裂解經轉染的細胞，並將此些內容物置入反應管中。研究人員們將一種合適的酶作用物，諸如使蟲螢光素酶能催化，產生光之化學反應的蟲螢光素，添加到反應管中。

A luminometer can detect and quantify the amount of light produced in each reaction tube.  The light intensity indicates the amount of target gene expression. If a protein upregulates target gene transcription, the cell expresses more luciferase molecules, which generates bright light. However, if the protein downregulates transcription, negligible luciferase expression occurs.

光度計能偵測及量化，於每一反應管中，產生的光量。此光強度顯示標的基因的表現量。倘若蛋白質上調標的基因轉錄，此細胞使更多產生明亮光的蟲螢光素酶分子，作出表現。不過，倘若蛋白質下調轉錄，則發生可忽略的蟲螢光素酶表現。

Promoter analysis in bacteria or eukaryotic cells typically employs a single luciferase reporter assay. This assay requires an excess of luciferin and ATP. A single luciferase reporter assay follows the classical methodology where scientists transfect cells with a promoter-reporter construct, followed by lysis, substrate introduction, and detection. Besides gene expression analysis, this assay is suitable for high throughput screening of protein-protein interactions as well.

於細菌或真核細胞之細胞中的啟動子分析，通常採用單一蟲螢光素酶信息測定。這種測定需要額外的蟲螢光素及三磷酸腺苷(ATP)。單一蟲螢光素酶信息測定遵循古典方法，在此科學家們使用啟動子與信息的構成物來轉染細胞，然後進行裂解、引入酶作用物及偵測。除了基因表現分析之外，該測定也適合，蛋白質與蛋白質之交互作用的高處理量篩選。

Scientists designed the split luciferase complementation (SLC) method to study protein-protein interactions.  This assay determines the effect of the physical contact between signaling proteins. The SLC method detects the factor that inhibits protein-protein interactions.

科學家們設計了，分裂蟲螢光素酶互補(SLC)法，來研究蛋白質與蛋白質的交互作用。此測定確定了，發信號蛋白質之間的物理接觸效果。該SLC方法發現了，抑制蛋白質與蛋白質之交互作用的因子。

It involves the genetic fusion of two inactive luciferase fragments with interacting proteins. The strongest luminescence corresponds to intense interactions between the protein pair and vice versa.

它涉及了，兩個不活躍之蟲螢光素酶片段與交互作用的蛋白質之遺傳因子融合。最強的發光對應於，在蛋白質對之間的強烈交互作用，且反之亦然。

Dual luciferase assays exploit the varying biochemical luminescence requirements of different species’ luciferase proteins. They allow the sequential quantitative measurement of FLuc and RLuc activities in a single protein extract.

雙重的蟲螢光素酶測定利用了，不同物種之蟲螢光素酶蛋白質的不同生化發光必需物。它們允許FLuc及RLuc活性，於單一蛋白質萃取物中的連續定量測量。

Scientists use this method to simultaneously monitor different cellular events and their regulation based on two distinct signal emissions (e.g., green and red light). The improvement in DLR applicability relies on the availability of a luminometer equipped with two optical filters.

科學家們使用此方法來同時監測，不同的細胞活動，及其根據兩種不同信號發出(譬如綠光及紅光)的調節。此在雙重-蟲螢光素酶之信息(DLR：Dual-Luciferase Reporter)適用性的改善，依賴配備兩個濾光器之光度計的可用性。

For example, researchers developed a DLR assay using two reporter genes (FLuc and RLuc) that quantifies sequential gene expression in yeast (Saccharomyces cerevisiae) using an advanced luminometer with wavelength-specific filters offering exceptional sensitivity for detecting distinct emissions.

譬如，研究人員們開發了一種，使用兩種信息基因(FLuc及RLuc)的DLR測定方法。該測定方法使用一種，具有提供偵測不同光發出特定波長之濾光器的先進光度計，來量化酵母菌(釀酒酵母)中，連續的基因表現。

Multicolor luciferase reporter assays are powerful molecular biological tools that enable simultaneous monitoring of multiple genes with a single substrate. Scientists designed reporter genes for multicolor luciferase assays using three separate luciferase genes that emit green, orange, and red light upon addition of a single substrate.

多色的蟲螢光素酶信息測定是一種，使以單一酶作用物能同時監測，多個基因之強有力的分子生物學工具。科學家們使用三種，在添加單一酶作用物，發出綠光、橙光及紅光之不同蟲螢光素酶基因，設計了用於多色蟲螢光素酶測定的信息基因。

This method’s major advantage is the ability to monitor sequential and complex expression changes in multiple genes within a single cell or tissue. Researchers apply this assay for high-throughput gene expression analysis.

此方法的主要優點是，監測單一細胞或組織內，於多個基因中，連續且複雜之表現變化的能耐。研究人員們將此測定法，應用於高處理量的基因表現分析。

A luciferase-substrate reaction emits light ranging across many wavelengths. The sensitivity of in vivo bioluminescence imaging depends on light emission at the NIR range (700-900nm) and tissue permeability of the number of photons emitted.

蟲螢光素酶與酶作用物的反應發出，橫跨多種波長的光。於活體內生物發光造影的靈敏度，取決於近紅外線(NIR：Near Infrared)範圍(700-900nm)發出的光及發出之光子數量的組織穿透性。

Scientists developed a NIR luciferin analog to detect photons from deep target tissues with high sensitivity. Currently, many researchers are focusing on tuning the color of light emitted by bioluminating reporter genes towards the NIR or IR region, which has better tissue penetrance.

科學家們開發了一種，類似偵測來自深層標的組織之光子，具高靈敏度的近紅外線蟲螢光素。目前，許多研究人員正著重於，將生物發光信息基因發出的光顏色，調整到具有更佳組織穿透性的近紅外線或紅外線區域。

Scientists are also working on multicomponent imaging to study several features or cell types simultaneously. The main concept behind this technique is the selective introduction of different luciferase molecules into separate cells within the same system. Upon specific substrate addition, only target cells light up.

科學家們也致力於多成分的造影，來同時研究多種特徵或細胞類型。此技術背後的主要概念是，將不同的蟲螢光素酶分子，選擇性引入相同物系內的不同細胞。於添加特定酶作用物後，僅標的細胞發光。

Scientists developed a multicolor bioluminescence system using NIR-emitting luciferases that enabled detection of two different cell populations within one animal model. A key shortcoming of this technique is the lack of a standardized method to visualize combinations of luciferase reporters. 

科學家們使用發出近紅外線的蟲螢光素酶開發了一種，使能偵測一種動物模型內，兩種不同細胞群的多色生物發光物系。此技術的一項主要缺點是，缺乏視化蟲螢光素酶信息組合的標準化方法。

網址：https://www.the-scientist.com/luciferase-a-powerful-bioluminescent-research-tool-72013

翻譯：許東榮