Firefly luciferase and NanoLuc luciferase double reporter gene detection kit

Abstract

The invention discloses a firefly luciferase and NanoLuc luciferase double reporter gene detection kit. The firefly luciferase and NanoLuc luciferase double reporter gene detection kit comprises a luciferase lysis solution, a firefly luciferase detection reagent, a NanoLuc luciferase detection buffer solution and a NanoLuc luciferase substrate (50 *). Tests show that the firefly luciferase and NanoLuc luciferase double-reporter gene detection kit prepared by the invention is high in detection sensitivity, wider in detection linear range, higher in signal, small in signal attenuation degree and slower in luminescence signal descending trend, and is more suitable for being applied to high-throughput detection.

Description

Technical Field

[0001] The present invention relates to biological detection technology, and in particular to a firefly luciferase and NanoLuc luciferase dual reporter gene detection kit. Background Art

[0002] Reporter gene refers to a group of genes that encode proteins or enzymes that are easily detected. The coding sequence of the reporter gene is fused with the gene expression regulatory sequence, or fused with other target genes, and expressed under the control of the regulatory sequence. The expression regulation of the target gene is "reported" by detecting the expression product of the reporter gene. Commonly used reporter genes include luciferase, chloramphenicol acetyltransferase (CAT), β-galactosidase (β-gal), secretory human placental alkaline phosphatase (SEAP) and green fluorescent protein (GFP). Among them, the luciferase reporter gene is widely used because of its simple analysis method, wide linear range, and low price.

[0003] The luciferase reporter gene is a reporter system that uses luciferin as a substrate to detect the activity of firefly luciferase. The principle is to construct the target gene transcriptional regulatory element into an expression vector with luciferase, and construct a reporter gene plasmid so that this sequence regulates the transcriptional expression of luciferase. Then the reporter gene plasmid is transfected into cells, and the cells are lysed after being given different treatments, and the substrate luciferin is added. Luciferase can catalyze luciferin to emit fluorescence. The fluorescence value obtained by the detection can determine the impact of different treatment groups on the transcriptional regulatory element.

[0004] Dual luciferase reporter genes, that is, two independent reporter genes are expressed and measured in one system, which is often used to improve experimental accuracy and avoid errors caused by differences in cell survival or plasmid transfection efficiency. Firefly and NanoLuc luciferase dual reporter genes are based on the firefly luciferase reporter gene and the NanoLuc luciferase reporter gene plasmid is simultaneously transferred into the firefly luciferase reporter gene as an internal reference to provide a reaction baseline, which is used as a correction parameter. Summary of the invention

[0005] In order to solve the need for detecting two independently expressed reporter genes in one system, the present invention proposes a detection kit for firefly and NanoLuc luciferase dual reporter genes, innovatively develops a NanoLuc luciferase detection buffer formula that effectively shields the fluorescent signal of firefly luciferase, and introduces the self-developed NanoLuc luciferase detection substrate (HFFz), thereby realizing high-throughput screening and use of dual luciferases with high light signal intensity, long duration and high sensitivity.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a firefly luciferase and NanoLuc luciferase dual reporter gene detection kit, comprising a luciferase lysate, a firefly luciferase detection reagent, a NanoLuc luciferase detection buffer and a NanoLuc luciferase substrate (50×).

[0008] The luciferase lysate comprises PBS buffer, nonionic surfactant, metal ion shielding agent, reducing agent, glycerol, bovine serum albumin, trehalose and sucrose. The luciferase lysate used in the kit of the present invention has the characteristics of mild cell lysis, protection of luciferase activity obtained by lysis, easy operation and no need for lysis on ice, and comprises stabilizers and carbohydrate protective agents, which further increases the stability of the enzyme.

[0009] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0010] Furthermore, the nonionic surfactant includes one of TritonX-100 and NP-40, and the volume fraction in the luciferase lysate is 0.1%-0.25%, preferably 0.1%. The nonionic surfactant can destroy the cell lipid bilayer and dissolve the cytoplasm and cell membrane.

[0011] Furthermore, the metal ion shielding agent includes ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, the content of which in the luciferase lysate is 3-5 mM, preferably 3 mM, and can shield metal ions under certain acidity conditions.

[0012] Furthermore, the reducing agent includes at least one of DTT, L-ascorbic acid and reduced glutathione, and the content of the reducing agent in the luciferase lysate is 3-15 mM, preferably 3 mM. The reducing agent can protect the enzyme activity.

[0013] Furthermore, the volume fraction of the glycerol in the luciferase lysate is 10%-15%, preferably 10%, and the glycerol can act as a stabilizer.

[0014] Furthermore, the mass volume ratio of the bovine serum albumin in the luciferase lysate is 0.1%-0.2%, preferably 0.15%, and the bovine serum albumin can act as a stabilizer.

[0015] Furthermore, the content of trehalose in the luciferase lysate is 10 mM, and trehalose can act as an enzyme protectant.

[0016] Furthermore, the content of sucrose in the luciferase lysate is 5 mM, and sucrose can act as an enzyme protectant.

[0017] The firefly luciferase detection reagent includes PBS buffer, ascorbic acid, ATP, magnesium chloride hexahydrate, D-luciferin potassium salt, and a reducing agent. The firefly luciferase detection reagent used in the kit of the present invention allows firefly luciferase to catalyze the oxidation of luciferin under the conditions of the simultaneous presence of detection substrates, oxygen, ATP, and magnesium ions, generating oxidized luciferin and yellow-green light, and the fluorescence brightness is proportional to the amount of luciferase. It has the characteristics of high sensitivity, wide linear range, fast and simple, and is particularly suitable for detection and high-throughput screening applications in aspects such as low cell signals, weak drug effects, and multi-cell interaction signals.

[0018] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0019] Furthermore, the content of ascorbic acid in the firefly luciferase detection reagent is 2-4 mM, preferably 2 mM. Ascorbic acid can stabilize and protect the luciferin substrate.

[0020] Furthermore, the content of ATP in the firefly luciferase detection reagent is 1-5 mM, preferably 5 mM. When ATP is hydrolyzed, the energy released in the PBS buffer is used as the energy source for the enzyme catalytic reaction.

[0021] Furthermore, the content of magnesium chloride hexahydrate in the firefly luciferase detection reagent is 1-5 mM, preferably 3 mM. The magnesium ions provided by magnesium chloride hexahydrate are essential ions for the reaction between firefly luciferase and substrate.

[0022] Furthermore, the content of D-luciferin potassium salt in the firefly luciferase detection reagent is 0.1-0.5 mg / mL, preferably 0.3 mg / mL, as a firefly luciferase substrate.

[0023] Furthermore, the reducing agent is reduced glutathione, and its content in the firefly luciferase detection reagent is 2-5 mM, preferably 2 mM, which plays a role in stabilizing and protecting the luciferin substrate.

[0024] The NanoLuc luciferase detection buffer contains PBS buffer, pyrophosphate, and EDTA. The NanoLuc luciferase detection buffer used in the kit of the present invention can effectively shield the fluorescent signal of firefly luciferase, with an inhibition rate of 99%, avoiding the influence of the fluorescent signal of firefly luciferase on the subsequent NanoLuc luciferase detection. After being prepared into the NanoLuc luciferase detection working solution with NanoLuc luciferase substrate (50×), the activity of NanoLuc luciferase can be detected without affecting the activity of NanoLuc luciferase.

[0025] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0026] Furthermore, the content of pyrophosphate in the NanoLuc luciferase assay buffer is 30-50 mg / mL, preferably 35 mg / mL.

[0027] Furthermore, the content of EDTA in the NanoLuc luciferase detection buffer is 8-12 mM, preferably 10 mM.

[0028] The NanoLuc luciferase substrate (50×) is HFFz, and DMSO is used as a solvent to dissolve HFFz to a content of 0.6-0.8 mg / mL, preferably 0.75 mg / mL. The NanoLuc luciferase substrate (50×) used in the kit of the present invention contains a novel substrate HFFz (Hydroxy lfl uorofur imaz ine) for detecting NanoLuc luciferase, which makes the light signal intensity large, the duration long, and the sensitivity high during the detection process. The NanoLuc luciferase signal can be detected by diluting the NanoLuc luciferase substrate (50×) 50 times with the NanoLuc luciferase detection buffer.

[0029] Furthermore, the preparation method of the substrate HFFz is described in Chinese invention patent application CN117924295A.

[0030] In a second aspect, the present invention provides the use of the firefly luciferase and NanoLuc luciferase dual reporter gene detection kit as described in the first aspect in improving the sensitivity of firefly luciferase reporter gene detection and NanoLuc luciferase reporter gene detection.

[0031] Firefly luciferase can catalyze luciferin to produce bioluminescent reaction. The reaction process is as follows:

[0032]

[0033] NanoLuc luciferase can catalyze the new substrate HFFz to produce a bioluminescent reaction. The reaction process is as follows:

[0034]

[0035] The present invention discloses a biological activity detection method, which comprises the following steps:

[0036] (1) Lysis of cells

[0037] (1.1) After aspirating the cell culture medium of cells transfected with firefly luciferase and NanoLuc luciferase reporter genes, add 1 mL of 0.25% trypsin to digest the cells, then add 5 mL of culture medium to resuspend the cells and count them. Aspirate the required number of cells and collect the cells by centrifugation. After removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E6 cells / mL and incubate at room temperature for 5 minutes to fully lyse the cells.

[0038] (1.2) After sufficient lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant for determination.

[0039] (2) Firefly luciferase detection

[0040] (2.1) Take 20 μL of supernatant and add it to the 96-well plate to be tested. Add 100 μL of firefly luciferase detection reagent, shake the plate to mix, and detect the activity of firefly luciferase.

[0041] (3) NanoLuc luciferase assay

[0042] (3.1) According to the amount of 100 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and dilute NanoLuc luciferase substrate (50×) 50 times to prepare NanoLuc luciferase assay working solution;

[0043] (3.2) Add 100 μL of NanoLuc luciferase assay working solution to the 96-well plate to be tested in step (2.1), shake the plate to mix, and detect the activity of NanoLuc luciferase.

[0044] Compared with the prior art, the beneficial effects of this application are:

[0045] The NanoLuc luciferase detection buffer of the present invention can effectively shield the fluorescent signal of firefly luciferase, with an inhibition rate of 99%, avoiding the influence of the fluorescent signal of firefly luciferase on subsequent NanoLuc detection. The present invention detects firefly luciferase by using D-luciferin potassium salt as a substrate, and innovatively introduces a new substrate HFFz (Hydroxyl fluorofur imazine) for detecting NanoLuc luciferase, so that the light signal has the characteristics of high intensity, long duration and high sensitivity during the detection process, which is conducive to high-throughput screening and use of dual luciferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the packaging of the four components of the kit.

[0047] Figure 2 Schematic diagram of the kit's results for detecting reporter gene activity.

[0048] Figure 3 This is a comparison chart of the linear performance test results of the kit for detecting firefly luciferase.

[0049] Figure 4 This is a comparison chart of the linear performance test results of the kit for detecting NanoLuc luciferase.

[0050] Figure 5 This is a comparison chart of the signal stability test results of the kit for detecting firefly luciferase.

[0051] Figure 6 This is a comparison chart of the signal stability test results of NanoLuc luciferase detected by the kit.

[0052] Figure 7 This is a comparison chart of the effects of different inhibitors on the inhibition rate of firefly luciferase.

[0053] Figure 8 This is a comparison chart of the effects of different NanoLuc substrates on the luminescent signal. DETAILED DESCRIPTION

[0054] The technical solutions in the embodiments are described clearly and completely below. Obviously, the embodiments described below are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0056] It should also be understood that the terms used in this description of the embodiments of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. As used in the description of the embodiments of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms of "a", "an" and "the" are intended to include plural forms.

[0057] The preparation method of the HFFz substrate involved in the present invention is described in Chinese invention patent application CN117924295A, and the preparation steps are as follows:

[0058] (1) p-Toluenesulfonyl azide and sodium hydride are dissolved in ultra-dry tetrahydrofuran as raw materials, stirred under ice bath conditions, and then diethylphosphonic acid tert-butyl ester is added, and the mixture is reacted under ice bath conditions for 20 to 40 minutes. The mixture is then transferred to room temperature and stirred for reaction for 1.5 to 2.5 hours. After the reaction is terminated, ethyl acetate is added for extraction, and the mixture is dried, filtered, and spin-dried to obtain intermediate 1, wherein the molar ratio of p-toluenesulfonyl azide, sodium hydride, and diethylphosphonic acid tert-butyl ester is 1.2:1:1;

[0059] (2) suspending zinc powder and iodine in anhydrous tetrahydrofuran and dry DMF, stirring at room temperature until the iodine fades, adding 2-fluorobenzyl bromide to react, cooling the reaction mixture to room temperature after the reaction, adding 2-amino-3,5-dibromopyrazine, PdCl2(PPh3)2, anhydrous tetrahydrofuran and dry DMF to stir and react, then filtering the mixture, extracting the filtrate, drying, concentrating and purifying to obtain intermediate 2; wherein the molar ratio of zinc powder, iodine, 2-fluorobenzyl bromide, 2-amino-3,5-dibromopyrazine and PdCl2(PPh3)2 is 3.8:0.38:1.5:1:0.05;

[0060] (3) Under an inert atmosphere, intermediate 1, intermediate 2, and dimer rhodium acetate are dissolved in toluene for reaction, and after cooling, extraction, washing, drying, concentration, and purification are performed to obtain intermediate 3, wherein the molar ratio of intermediate 2 to intermediate 1 and dimer rhodium acetate is 1:1 to 2:0.1, the reaction temperature is 100 to 110° C., and the reaction time is 22 to 26 hours;

[0061] (4) intermediate 3 and 2-furfural are dissolved in methanol, tetramethylguanidine is added for reaction, and intermediate 4 is obtained after extraction, drying, concentration and purification; wherein the molar ratio of intermediate 3, 2-furfural and tetramethylguanidine is 1:1-2:2-3; the reaction temperature is room temperature, and the reaction time is 1.5-3.5h;

[0062] (5) Under an inert atmosphere, the intermediate 4 is dissolved in a 1,4-dioxane solution, and tetrakistriphenylphosphine palladium, a phenylboric acid compound, and a cesium carbonate aqueous solution are added, and the reaction is carried out at 85° C. for 3 to 4 hours. After cooling, extraction, drying, concentration, and purification are performed to obtain an intermediate 5, wherein the molar ratio of the intermediate 4, tetrakistriphenylphosphine palladium, a phenylboric acid compound, and a cesium carbonate aqueous solution is 1:0.1:1.3:3; in the 1,4-dioxane solution, the volume ratio of 1,4-dioxane to water is 5:1; and the phenylboric acid compound is 3-(tert-butyldimethylsilyloxy)-2-fluorophenylboric acid;

[0063] (6) Intermediate 5 was dissolved in dichloromethane and then placed in an ice bath, and trifluoroacetic acid was added. The reaction was stirred at room temperature for 4 to 6 hours, and the volatiles were removed under reduced pressure to obtain intermediate 6, wherein the molar ratio of intermediate 5 to trifluoroacetic acid was 1:9 to 10;

[0064] (7) Intermediate 6 is dissolved in dichloromethane, and then CDI is added, and the reaction is carried out in an ice bath for 0.5-2.5 hours. After extraction, drying, concentration and purification, intermediate 7 is obtained, wherein the molar ratio of intermediate 6 to CDI is 1:1.5-2.5;

[0065] (8) Dissolving intermediate 7 in dichloromethane and methanol, adding sodium borohydride after cooling and reacting for 20 to 40 minutes, adding hydrochloric acid to the reaction solution, adjusting the pH, extracting, drying, concentrating and purifying to obtain intermediate 8, wherein the molar ratio of intermediate 7 to sodium borohydride is 1:2 to 4, and the concentration of hydrochloric acid is 0.1 mol / L;

[0066] (9) The intermediate 8 is dissolved in methanol, hydrochloric acid is added, and the reaction is carried out for 16 to 24 hours. HFFz is obtained after extraction, drying, concentration and purification, wherein the concentration of hydrochloric acid is 6 mol / L, and the molar ratio of the intermediate 8 to the hydrochloric acid is 1:8 to 20.

[0067] Other raw materials involved in the present invention can be directly purchased from the market. For process parameters not specifically noted, conventional techniques can be used.

[0068] like Figure 1 As shown in the figure, the kit consists of 4 packaging bottles, and the contents of the packaging bottles are luciferase lysate, firefly luciferase detection reagent, NanoLuciferase detection buffer and NanoLuciferase substrate (50×) from left to right.

[0069] Example 1

[0070] This embodiment provides a firefly luciferase and NanoLuc luciferase dual reporter gene detection kit, which includes four reagents: luciferase lysate, firefly luciferase detection reagent, NanoLuc luciferase detection buffer and NanoLuc luciferase substrate (50×), and the raw material composition and preparation method of each reagent are as follows.

[0071] (1) Preparation of luciferase lysate

[0072] In this embodiment, the luciferase lysis solution includes PBS buffer, non-ionic surfactant, metal ion shielding agent, reducing agent, glycerol, bovine serum albumin, trehalose, and sucrose.

[0073] The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4; the nonionic surfactant is NP-40, the metal ion shielding agent is ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, and the reducing agents are DTT and reduced glutathione.

[0074] 40 mL of PBS buffer was measured and put into a container, and then 0.1% volume fraction of NP-40, 3 mM of ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, 3 mM of DTT, 3 mM of reduced glutathione, 10% volume fraction of glycerol, 0.15% mass volume ratio of bovine serum albumin, 10 mM of trehalose, 5 mM of sucrose were added, and finally the volume was made up to 50 ml with PBS buffer. The above components can be dissolved by conventional stirring method, and the obtained transparent viscous liquid is the luciferase lysate.

[0075] (2) Preparation of firefly luciferase detection reagent

[0076] In this embodiment, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, the reducing agent is reduced glutathione, and also includes ascorbic acid, ATP, magnesium chloride hexahydrate, and D-luciferin potassium salt.

[0077] Measure 40 mL of PBS buffer into a container, then add 3 mM reduced glutathione, 2 mM ascorbic acid, 5 mM ATP, 3 mM magnesium chloride hexahydrate, and 0.3 mg / mL D-luciferin potassium salt, and finally dilute to 50 ml with PBS buffer. The resulting light yellow liquid is the firefly luciferase detection reagent.

[0078] (3) Preparation of NanoLuc luciferase assay buffer

[0079] Measure 40 mL of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 35 mg / mL pyrophosphate and 10 mM EDTA. Finally, dilute to 50 ml with PBS buffer. The resulting transparent liquid is the NanoLuc luciferase detection buffer.

[0080] (4) Preparation of NanoLuc luciferase substrate (50×)

[0081] 10 ml of DMSO was measured and added to the container, and HFFz was weighed to make its concentration 0.65 mg / mL, which is the NanoLuc detection substrate (50×).

[0082] Example 2

[0083] This embodiment provides a firefly luciferase and NanoLuc luciferase dual reporter gene detection kit, which includes four reagents: luciferase lysate, firefly luciferase detection reagent, NanoLuc luciferase detection buffer and NanoLuc luciferase substrate (50×), and the raw material composition and preparation method of each reagent are as follows.

[0084] (1) Preparation of luciferase lysate

[0085] In this embodiment, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, the non-ionic surfactant is NP-40, the metal ion shielding agent is ethylene glycol bis(2-aminoethyl ether)tetraacetic acid, the reducing agent is DTT and reduced glutathione, and the rest are glycerol, bovine serum albumin, trehalose, and sucrose.

[0086] 40 mL of PBS buffer was measured into a container, and then 0.2% volume fraction of NP-40, 5 mM of ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, 5 mM of DTT, 5 mM of reduced glutathione, 15% volume fraction of glycerol, 0.2% mass volume ratio of bovine serum albumin, 10 mM of trehalose, and 5 mM of sucrose were added. Finally, the volume was made up to 50 mL with PBS buffer. The above components can be dissolved by conventional stirring method, and the obtained transparent viscous liquid is the luciferase lysate.

[0087] (2) Preparation of firefly luciferase detection reagent

[0088] In this embodiment, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4, the reducing agent is reduced glutathione, and also includes ascorbic acid, ATP, magnesium chloride hexahydrate, and D-luciferin potassium salt.

[0089] Measure 40 mL of PBS buffer into a container, then add 5 mM reduced glutathione, 4 mM ascorbic acid, 3 mM ATP, 5 mM magnesium chloride hexahydrate, and 0.5 mg / mL D-luciferin potassium salt, and finally dilute to 50 mL with PBS buffer. The resulting light yellow liquid is the firefly luciferase detection reagent.

[0090] (3) Preparation of NanoLuc luciferase assay buffer

[0091] Measure 40 mL of PBS buffer into a container. The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4. Then add 50 mg / mL pyrophosphate and 12 mM EDTA. Finally, dilute to 50 mL with PBS buffer. The resulting transparent liquid is the NanoLuc luciferase detection buffer.

[0092] (4) Preparation of NanoLuc luciferase substrate (50×)

[0093] 10 mL of DMSO was measured and added to the container, and HFFz was weighed to make its concentration 0.75 mg / mL, which is the NanoLuc detection substrate (50×).

[0094] Comparative Example 1:

[0095] A commercially available A-type kit (Nano & Firefly-Glo Dual Luciferase Reporter Gene Assay Kit, Meilun, MA0522-1) was used.

[0096] Comparative Example 2

[0097] The kit of this comparative example replaces the pyrophosphate component in the NanoLuc luciferase detection buffer in Example 1 with the firefly luciferase inhibitor KI at a content of 40 mM. The other components and preparation method are consistent with those in Example 1.

[0098] Comparative Example 3

[0099] The kit of this comparative example replaces the pyrophosphate component in the NanoLuc luciferase detection buffer of Example 1 with the firefly luciferase inhibitor resveratrol at a content of 1 mM. Other components and preparation methods are consistent with those of Example 1.

[0100] Comparative Example 4

[0101] The kit of this comparative example replaces the HFFz in the NanoLuc luciferase substrate (50×) of Example 1 with FZ (Furimazine), a substrate of another NanoLuc luciferase, and the preparation method and concentration are consistent with those of Example 1.

[0102] Example 3

[0103] The kit reporter gene activity detection, the experimental steps are as follows:

[0104] 1. Detection method

[0105] (1) Lysis of cells

[0106] (1.1) After aspirating the cell culture medium of cells transfected with firefly luciferase and NanoLuc luciferase, add 1 mL of 0.25% trypsin to digest the cells, add 5 mL of culture medium to resuspend the cells and count them, aspirate the required number of cells, and collect the cells by centrifugation; after removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E6 cells / mL, and incubate at room temperature for 5 minutes to fully lyse the cells.

[0107] (1.2) After sufficient lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant for determination.

[0108] (2) Firefly luciferase detection

[0109] (2.1) Take 20 μL of supernatant and add it to the 96-well plate to be tested. Add 100 μL of firefly luciferase detection reagent, shake the plate to mix, and detect the activity of firefly luciferase.

[0110] (3) NanoLuc luciferase assay

[0111] (3.1) According to the amount of 100 μL NanoLuc luciferase assay working solution required for each sample, 294 μL NanoLuc luciferase assay buffer and 6 μL NanoLuc luciferase substrate (50×) were prepared to prepare NanoLuc luciferase assay working solution.

[0112] (3.3) Add 100 μl of NanoLuc luciferase detection working solution to the 96-well plate to which the firefly luciferase detection reagent has been added in step (2.1), shake the plate to mix, and detect the activity of NanoLuc luciferase.

[0113] The above test was performed on the kits prepared in Example 1, Example 2 and Comparative Example 1 for comparison.

[0114] The results are as follows Figure 2As shown, it is shown that the kit prepared in the present application has a higher detection sensitivity for firefly luciferase and NanoLuc luciferase than conventional commercially available kits.

[0115] Example 4

[0116] The test kit linear performance test steps are as follows:

[0117] In this example, the pure enzyme solution containing firefly luciferase and NanoLuc luciferase was diluted with luciferase lysate to simulate the enzyme expression of firefly luciferase and NanoLuc dual luciferase reporter genes in cells. The test results were linearly fitted with a curve of enzyme concentration and detection signal value (RLU).

[0118] 1. Kit detection method

[0119] (1) Take luciferase lysis buffer and dilute pure firefly luciferase enzyme (brand: Besty, model: P045) to a concentration of 1 μg / mL, mix well, and then continue to use the above lysis buffer containing firefly luciferase to dilute pure NanoLuc luciferase enzyme (brand: Besty, model: P1412) to a concentration of 100 ng / mL, and then continue to use the above lysis buffer containing two luciferases to dilute the above lysis buffer with luciferase lysis buffer to 6 concentrations. After dilution, the luciferase The enzyme lysate contained firefly luciferase at concentrations of 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.062 μg / mL, 0.031 μg / mL, and 0.015 μg / mL, while the luciferase lysate contained NanoLuc luciferase at concentrations of 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.1 ng / mL, and 1.56 ng / mL.

[0120] (2) Take 20 μL of the above luciferase lysate with different enzyme concentrations and add it to the 96-well plate to be tested, add 100 μL of firefly luciferase detection reagent to each well, shake the plate to mix, and use a multifunctional microplate reader (Berthokd LB941) to detect the activity of firefly luciferase.

[0121] (3) According to the amount of 100 μL NanoLuc luciferase assay working solution required for each sample, 588 μL NanoLuc luciferase assay buffer and 12 μL NanoLuc luciferase substrate (50×) were prepared to prepare NanoLuc luciferase assay working solution.

[0122] (3.3) Add 100 μl of NanoLuc luciferase detection working solution to each well of the 96-well plate to which the firefly luciferase detection reagent has been added in step (2), shake the plate to mix well, and use a multi-function microplate reader (Berthokd LB941) to detect the activity of NanoLuc luciferase.

[0123] The above test was carried out using the kits prepared in Example 1, Example 2 and Comparative Example 1 for comparison.

[0124] The results of the linear test of firefly luciferase are as follows Figure 3 As shown, it is shown that the kit prepared in the present application has a wider linear range for detecting firefly luciferase and a higher signal compared with conventional commercially available kits.

[0125] The results of the NanoLuc luciferase linearity test are as follows Figure 4 As shown, it is shown that the kit prepared in the present application has a wider linear detection range for NanoLuc luciferase and a higher signal compared with conventional commercially available kits.

[0126] Example 5 Kit Signal Stability Detection

[0127] 1. Detection of Firefly Luciferase Signal Stability

[0128] (1) Take luciferase lysate and dilute pure firefly luciferase enzyme (brand: Besty, model: P045) to a concentration of 0.5 μg / mL.

[0129] (2) Take 20 μL of luciferase lysate containing 0.5 μg / mL firefly luciferase and add it to the 96-well plate to be tested, add 100 μL of firefly luciferase detection reagent, make three replicate wells, shake the plate to mix, use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal, set it to measure the fluorescence value every 5 minutes, measure the fluorescence value for a total of 30 minutes, and read the average fluorescence value at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min for subsequent analysis.

[0130] 2. Detection of NanoLuc luciferase signal stability

[0131] (1) Take NanoLuc luciferase pure enzyme (brand: Besty, model: P1412) and dilute it with luciferase lysis buffer to a concentration of 50 ng / mL.

[0132] (2) According to the amount of 100 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and dilute the NanoLuc luciferase substrate (50×) 50 times to prepare the NanoLuc luciferase assay working solution.

[0133] (3) Take 20 μL of luciferase lysate containing 50 ng / mL NanoLuc luciferase and add it to the 96-well plate to be tested, add 100 μL NanoLuc luciferase detection working solution, make 3 replicate wells, shake the plate to mix, use a multi-function microplate reader (Berthokd LB941) to detect the chemiluminescent signal, set it to measure the fluorescence value every 5 minutes, measure the fluorescence value for a total of 30 minutes, and read the average fluorescence value at 0min, 5min, 10min, 15min, 20min, 25min, and 30min for subsequent analysis.

[0134] The above test was performed on Example 1, Example 2 and Comparative Example 1 for comparison.

[0135] The luminescence stability results of firefly luciferase detection are as follows Figure 5 As shown, it is shown that the signal of the kit of the present invention decreases slowly within 30 minutes. Compared with Comparative Example 1, the signal attenuation degree of the kit of the present invention within 30 minutes is small.

[0136] The luminescence stability results of NanoLuc luciferase assay are as follows Figure 6 As shown, it is shown that the signal of the kit of the present invention decreases slowly within 30 minutes. Compared with Comparative Example 1, the signal attenuation degree of the kit of the present invention within 30 minutes is small.

[0137] Example 6 Effects of different inhibitors on the inhibition rate of firefly luciferase

[0138] The experimental steps are as follows:

[0139] 1. Detection method

[0140] (1) Lysis of cells

[0141] (1.1) After aspirating the cell culture medium of cells transfected with firefly luciferase and NanoLuc luciferase, add 1 mL of 0.25% trypsin to digest the cells, add 5 mL of culture medium to resuspend the cells and count them, aspirate the required number of cells, and collect the cells by centrifugation; after removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E6 cells / mL, and incubate at room temperature for 5 minutes to fully lyse the cells.

[0142] (1.2) After sufficient lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant for determination.

[0143] (2) Firefly luciferase detection

[0144] (2.1) Take 20 μL of supernatant and add it to the 96-well plate to be tested. Add 100 μL of firefly luciferase detection reagent, shake the plate to mix, and detect the activity of firefly luciferase.

[0145] (3) NanoLuc luciferase assay

[0146] (3.1) In the 96-well plate of step (2.1), add 98 μL of NanoLuc luciferase detection buffer to each well, shake the plate to mix, and detect the chemiluminescent signal. The obtained signal value reflects the inhibitory effect of the buffer on the firefly luciferase catalytic reaction in the previous step.

[0147] (3.2) Add 2 μL of NanoLuc luciferase substrate (50×) to the 96-well plate in step (3.1), shake the plate to mix, and use a multi-function microplate reader (Berthokd LB941) to detect the activity of NanoLuc luciferase.

[0148] The above test was carried out using Example 1, Example 2 and Comparative Example 2, Comparative Example 3.

[0149] The results are as follows Figure 7 As shown, it is shown that the inhibitory effect of pyrophosphate on firefly luciferase is more advantageous than the inhibitory effect of conventional KI and resveratrol, and has no inhibitory effect on NanoLuc luciferase and does not affect the subsequent chemiluminescent signal determination.

[0150] Example 7 Effects of different NanoLuc luciferase substrates on luminescent signals

[0151] (1) According to the amount of 100 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and dilute NanoLuc luciferase substrate (50×) 50 times to prepare NanoLuc luciferase assay working solution.

[0152] (2) Take 20 μL of lysate containing 50 ng / mL NanoLuc luciferase and add it to the 96-well plate to be tested, add 100 μL NanoLuc luciferase detection working solution, make 3 replicate wells, shake the plate to mix, use a multi-function microplate reader (Berthokd LB941) to detect the chemiluminescent signal, set it to measure the fluorescence value every 5 minutes, measure the fluorescence value for a total of 30 minutes, and read the average fluorescence value at 0min, 5min, 10min, 15min, 20min, 25min, and 30min for subsequent analysis.

[0153] The above test was performed on Example 1 and Comparative Example 4 for comparison.

[0154] The results are as follows Figure 8 As shown, the decreasing trend of the luminescent signal of the novel substrate HFFz within 30 minutes of detection is slower than that of the conventional substrate FZ, indicating that the substrate used in the present invention is more suitable for the application of the kit in high-throughput detection.

[0155] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A firefly luciferase and NanoLuc luciferase dual reporter gene detection kit, characterized in that: Includes Luciferase Lysis Buffer, Firefly Luciferase Assay Reagent, NanoLuciferase Assay Buffer, and NanoLuciferase Substrate (50×).

2. The kit according to claim 1, characterized in that The luciferase lysis solution comprises PBS buffer, nonionic surfactant, metal ion shielding agent, reducing agent, glycerol, bovine serum albumin, trehalose and sucrose.

3. The kit according to claim 2, characterized in that The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4; The non-ionic surfactant includes one of TritonX-100 and NP-40, and the volume fraction in the luciferase lysate is 0.1%-0.25%; The metal ion shielding agent includes ethylene glycol bis(2-aminoethyl ether) tetraacetic acid, and the content in the luciferase lysate is 3-5 mM; The reducing agent includes at least one of DTT, L-ascorbic acid, and reduced glutathione, and the content of the reducing agent in the luciferase lysate is 3-15 mM; The volume fraction of the glycerol in the luciferase lysate is 10%-15%; The mass volume ratio of the bovine serum albumin in the luciferase lysate is 0.1%-0.2%; The content of trehalose in the luciferase lysate is 10 mM; The content of sucrose in the luciferase lysate is 5 mM.

4. The kit according to claim 1, characterized in that The firefly luciferase detection reagent comprises PBS buffer, ascorbic acid, ATP, magnesium chloride hexahydrate, D-luciferin potassium salt and a reducing agent.

5. The kit according to claim 4, characterized in that The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4; The content of ascorbic acid in the firefly luciferase detection reagent is 2-4 mM; The content of ATP in the firefly luciferase detection reagent is 1-5mM; The content of magnesium chloride hexahydrate in the firefly luciferase detection reagent is 1-5mM; The content of D-luciferin potassium salt in the firefly luciferase detection reagent is 0.1-0.5 mg / mL; The reducing agent is reduced glutathione, and its content in the firefly luciferase detection reagent is 2-5 mM.

6. The kit according to claim 1, characterized in that The NanoLuc luciferase detection buffer contains PBS buffer, pyrophosphate, and EDTA.

7. The kit according to claim 6, characterized in that The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

8. The kit according to claim 6, characterized in that The content of pyrophosphate in the NanoLuc luciferase assay buffer is 30-50 mg / mL.

9. The kit according to claim 6, characterized in that The content of EDTA in the NanoLuc luciferase assay buffer is 8-12 mM.

10. The kit according to claim 1, characterized in that The NanoLuc luciferase substrate (50×) is HFFz, and the content is 0.6-0.8 mg / mL.

11. Use of the firefly luciferase and NanoLuc luciferase dual reporter gene detection kit according to any one of claims 1 to 10 in improving the sensitivity of firefly luciferase reporter gene detection and NanoLuc luciferase reporter gene detection.

Images