Fluorescein +ATP → fluorescein adenylate) +PPi
Fluorescein adenylate +O2 → oxyfluorescein +AMP+ light
This reaction is very energy-saving, and almost all the energy input into the reaction is converted into light. In sharp contrast to incandescent lamps used by human beings, only more than 10% of energy is converted into light, and the rest energy is converted into heat energy and wasted.
Luciferin or luciferase is not a specific molecule, but a general term for all substrates that can produce fluorescence and their corresponding enzymes, although they are different. Different organisms that can control luminescence use different luciferases to catalyze different luminescence reactions. The most well-known luminescent organisms are fireflies, and the luciferases they use are different from those of other luminescent organisms, such as Pleurotus ostreatus or many marine organisms. In fireflies, the oxygen needed for luminescence reaction is input from a tube called abdominal trachea. Some organisms, such as the insect, contain many different luciferases, which can catalyze the same luciferase substrate and emit different colors of fluorescence. There are more than 2000 kinds of fireflies. However, there are many other insects (including fireflies, kowtowing insects and related insects) in ladybug superfamily, so their luciferases are very useful for molecular systematics. At present, the most thoroughly studied luciferase is the North American firefly (Photinus pyralis) from Photinini firefly family.
luciferase
Also known as luminescent enzyme, it is the general name of enzyme system that catalyzes bioluminescence. It is a high molecular component, which is unstable to heat after the cold water extract of light substances glows in oxygen and the substrate fluorescein is consumed. At present, the most studied are fireflies and luminescent bacteria fluorescein crystals. They belong to oxygenase, and contain no metal and coenzyme. In order to emit light, some enzymes must use ATP as an auxiliary factor. No one else did. As we all know, the luminous mechanism of fireflies varies greatly from species to species. Luciferase is highly specific and usually only acts on luciferases of related species. Of course, fireflies and firefly enzymes can't replace each other to cause luminescence. Luciferase from fireflies is quite stable in dry state and can be preserved.
Double luciferase reporter gene detection: an advanced reporter gene detection technology combining fireflies and marine coelenterates luciferase.
When firefly luciferase is used to quantify gene expression, the second reporter gene is usually used to reduce the variation factors in the experiment. However, the traditional * * * reporter genes (such as CAT, β-Gal, GUS) are not convenient because of their different test chemistry, processing requirements and detection characteristics. Promega provides advanced dual-reporter gene technology. Combined with firefly luciferase test and marine coelenterate luciferase test, dual luciferase reporter gene detection system and pRL carrier system, the second reporter gene, marine coelenterate luciferase, was expressed in a single tube, which was rapid, sensitive and simple. The system also provides PLB lysate to lyse mammalian cells cultured in multi-well plates without manipulating a single sample. For researchers who use firefly luciferase reporter gene vectors ..
introduce
Double reporter genes are used for correlation or proportional detection in experimental systems. Usually, one reporter gene is used as an internal control, so that the detection of the other reporter gene is consistent. When gene expression is detected, double reporter genes are usually used to transiently transfect cultured cells, and a vector with experimental reporter genes is transfected with a second vector with different reporter genes as a control. Usually, the experimental reporter gene is coupled with a regulated promoter. To study the structure and physiological basis of regulatory genes. The relative change of reporter gene expression activity is related to the change of transcription activity of coupled regulatory promoter. The second reporter gene coupled with the constitutive promoter provides internal control of transcription activity, so the experiment is not disturbed by the change of experimental conditions.
By this method, the experimental accuracy weakened by internal factors, such as the difference in the number and vitality of cultured cells and the efficiency of cell transfection and lysis, can be reduced.
In recent years, firefly luciferase combined with chloramphenicol acetyltransferase (CAT), β -galactosidase (β-Gal) or glucuronidase (GUS) has been widely used. However, the combination of these double reporter genes weakens the advantages of luciferase operation. For example, luciferase test and quantification can be carried out in a few seconds, but the methods of CAT, β-Gal and GUS test, in addition, these reporter genes are limited by their sensitivity and linear response range, so we must be careful not to exceed these ranges, and endogenous cell vitality will also interfere with the use of such reporter genes. Many types of cells have endogenous β-Gal or GUS expression, which is not conducive to the accurate and quantitative expression of reporter genes. Intracellular deacetylase activity interferes with CAT activity test. Although high temperature pretreatment of cell lysate (1, 2) can reduce the interference of endogenous β-Gal and CAT tests, these treatments can also inactivate luciferase rapidly. Therefore, in this double reporter gene detection, it is necessary to treat the cell lysate transfected with * * * in different steps.
The ideal double reporter gene method should enable users to simultaneously determine two reporter genes in the same sample with the speed, sensitivity and linear range of firefly luciferase. This is impossible in traditional reporter genes, such as CAT, β-Gal and GUS, because of their inherent limitations in test chemistry. On the contrary, the dual luciferase reporter gene test (DLR) system of Promega, which combines firefly (Pyraris) and Renilla reniformis, can meet these requirements and complete these tests in a single test tube.
Double luciferase reporter gene test chemistry
Both fireflies and marine coelenterates have excellent test characteristics of bioluminescence reporter genes, but their evolutionary origins are different, so they have different enzyme structures and substrate requirements. These differences are used to develop DLR test chemistry and selectively distinguish the activities of these two bioluminescence reporter genes. Firefly luciferase is a protein with 6 1kDa subunit. Enzyme activity can be used as a genetic reporter gene after translation without post-translation modification (3, 4). In the presence of ATP, Mg 2+ and O 2, it emits light through the oxidation reaction of beetle fluorescein (Figure 1). Under normal reaction conditions, when fluorescein is oxidized, the conversion with fluorescein -AMP as the intermediate is very slow. As a result, after the substrate and enzyme are mixed, the test chemistry produces "flicker" light, which rapidly decays. The patented test reagent can quantify the activity of firefly luciferase, and coenzyme A(CoA) is added to enhance the rapid enzyme transformation, so as to improve the reaction kinetics (5), thus generating a continuous "blinking" luminous signal (Figure 2).
Fig. 1 bioluminescence catalyzed by fireflies and sea kidney luciferase
Marine coelenterase is a 36 kDa protein, which is purified from the natural kidney-shaped sea kidney (6) and contains 3% carbohydrate. But like firefly luciferase, its activity can be used as a genetic reporter gene without post-translation modification. The luminescence reaction catalyzed by marine coelenterate uses O _ 2 and coelenterate (Figure 1). When the DLR test chemistry is used in the experiment, the kinetics of the reaction of marine coelenterate produces a scintillation luminescence signal, which slowly decays during the detection process (Figure 2).
Luciferase activities of fireflies and marine coelenterates were measured step by step with a single lysate in DLR test system. After the activity of firefly luciferase ("experimental" reporter gene) was determined, the luminescence of firefly was quickly annihilated, and the luminescence reaction of marine coelenterate luciferase was activated ("control" reporter gene). Therefore, the DLR test system integrates two test chemicals to quickly quantify the two reporter genes expressed in * * *.
The linear range of firefly luciferase test extends to 8 orders of magnitude of enzyme concentration, and the experimental report gene enzyme ≤ 1fg (about 10 -20 mol) can be determined (Figure 3A). Using the double luciferase reporter gene detection system, the linear range of luciferase in marine coelenterates reached seven orders of magnitude, and the lower limit was ≤ 10fg.
Detection of luminescence produced by firefly and sea kidney luciferase by double luciferase reporter gene. CHO cells were transfected with pGL3 (1× 10 6 /60mm culture plate).
Control and pRL SV40 vector DNA. After washing the cells with PBS, 400μl PLB was added to prepare the lysate. 20μl of small cell lysate was mixed with 100μl of luciferase test reagent II(LARII), and the activity of firefly luciferase was immediately detected by fluorescence photometer (thin line tracing) 5438+000μ l of stop &; Glo TM reagent was added to the tube of the fluorometer to eliminate the firefly luciferase reaction and activate the renilla luciferase reaction at the same time, and the activity of renilla luciferase was immediately detected (thick line tracking). Turner Designs 20/20 fluorometer is equipped with a computer to track the fluorescence emission, and the test is completed twice in 65438 02 seconds.
Method of dual luciferase reporter gene detection system
Luminescent signals of luciferases from two reporter genes can be quantified immediately after the lysis solution is prepared, and it is not necessary to divide the samples into groups or carry out additional treatment. Because both marine coelenterates and fireflies show scintillation reaction kinetics, the dual luciferase reporter gene test does not need a fluorometer with a reagent syringe.
Typically, the DLR test takes about 30 seconds to complete, as shown in Figure 4. When the firefly luciferase reporter gene test was started, the cleavage product was mixed with luciferase test reagent II (LAR II). When the firefly luciferase test was completed, the firefly luminescence was annihilated, and at the same time, the luciferase luminescence of marine coelenterates was activated and stopped. Glo TM reagent to the sample tube. Within 1 sec, stop &; Glo TM reagent annihilates the luminous signal of firefly reaction with titer greater than 1.05 (Figure 5), and activates luciferase of marine coelenterates.
Fig. 3 Linear range of luminescence reaction between firefly luciferase and sea kidney luciferase. The purified firefly and renilla luciferase were continuously diluted with 1mg/ml BSA. Turner Designs fluorometer equipped with computer is used to detect the total fluorescence of 65438 00 seconds. After the pre-reading delay of the first 2 seconds, a neutral density filter was added to the sample room of the fluorometer when the luminescence reaction of two high-concentration luciferases was directly detected. In the luciferase reaction containing 10fg and 100fg, the background signal from the autoluminescence of luciferase in the ocean cavity was subtracted. The luminescent activities of the two luciferases were plotted against their respective test change concentrations.
Figure 4. Method of double luciferase test with manual fluorometer or fluorometer equipped with reagent sampler. For example, if the fluorometer is equipped with two samplers, the lysate is prepackaged into the tube of the fluorometer, and then luciferase assay reagent II (LAR II), stop &: GloTM reagent.
PLB lysate
PLB lysate buffer, specially designed, can effectively lyse cultured mammalian cells without scraping adherent cells or freeze-thaw cycle. Although PLB is designed for passive pyrolysis process, its reliable pyrolysis effect is also beneficial to the pyrolysis products prepared by conventional treatment methods. No matter what lysis method is used, fireflies and marine coelenterate enzyme reporter released into PLB lysate are quantitative and reliable for cultured mammalian cells (Figure 6). An obvious advantage of passive lysate is that it can inhibit low-level non-enzymatic luminescence (autoluminescence), which is an inherent feature of marine coelenterates in aqueous solution. The reagents commonly used to prepare cell lysate can enhance the autoluminescence of marine coelenterates, including Triton? X- 100, cell culture lysis test of Promega? (CCLR) and reporter gene cleavage reagent (RLB), which can also significantly inhibit the luminescent reaction of marine coelenterates luciferase. PLB is specially designed to maximize luciferase activity and minimize autoluminescence, which can provide the best test sensitivity and quantify very low levels of luciferase in marine coelenterates. In addition, PLB inhibits foaming, which is very suitable for Qualcomm application, and can be used in automatic systems to prepare cell populations cultured in porous plates into lysates and test them.
Fig. 5 Dual luciferase reporter gene test system eliminates firefly luciferase luminescence and activates renilla luciferase luminescence. CHO cell lysate contains * * * transfected pgl 3- control and pRL SV40 vector DNA, which was prepared according to the method described in Figure 2. The activities of firefly luciferase (reporter gene 1) and renilla luciferase (reporter gene 2) were detected within 10 second. Glo TM reagent annihilates the high efficiency of reporter gene 1, and adds equal volume of stop &; Glo TM reagent (without coelenteratin, the luciferase reaction can not be activated), firefly luciferase luminescence is annihilated without activating the luciferase luminescence. In this experiment, the residual luminescence of the destroyed firefly luciferase reaction is less than 0.0004% of the undamaged reaction value.