Firefly-NanoLuc-AkaLuc tri-luciferase reporter gene detection kit

Abstract

The invention discloses a firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit. The firefly-NanoLuc-AkaLuc three-luciferase reporter gene detection kit comprises a luciferase lysis solution, a firefly luciferase detection reagent, a NanoLuc luciferase detection buffer solution, a NanoLuc luciferase substrate concentrated solution, an AkaLuc luciferase detection buffer solution and an AkaLuc luciferase substrate concentrated solution. Tests show that the firefly-NanoLuc-AkaLuc triluciferase 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 light-emitting signal descending trend, and is more suitable for being applied to high-throughput detection.

Description

Technical Field

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

[0002] Reporter genes refer 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 transcriptional regulatory element of the target gene into an expression vector with luciferase to 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 different treatments are given, 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] Firefly luciferase, NanoLuc luciferase and AkaLuc luciferase can catalyze their respective substrates to emit fluorescence of different colors. The three light absorption wavelengths are different, and the detection does not interfere with each other. Therefore, they can be used simultaneously in the same chemical reaction system. This has become an effective means to study the involvement of transcription factors in gene regulation. Summary of the invention

[0005] In order to detect three independently expressed reporter genes in the same system, the present invention proposes a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit, and innovatively develops NanoLuc luciferase detection buffer and AkaLuc luciferase detection buffer formulas, wherein the NanoLuc luciferase detection buffer can effectively shield the fluorescence signal of firefly luciferase, and the AkaLuc luciferase detection buffer can effectively shield the fluorescence signal of NanoLuc luciferase, so that high-throughput screening and use with high light signal intensity, long duration and high sensitivity can be achieved in the fluorescence signal detection of firefly luciferase, NanoLuc luciferase and AkaLuc luciferase in sequence.

[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-NanoLuc-AkaLuc triple luciferase reporter gene detection kit, comprising luciferase lysate, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate.

[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 non-ionic 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 non-ionic 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 the NanoLuc luciferase substrate concentrate, 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 concentrate is HFFz, and DMSO is used as a solvent to dissolve HFFz so that its content is 0.6-0.8 mg / mL, preferably 0.75 mg / mL. The NanoLuc luciferase substrate concentrate used in the kit of the present invention contains a new substrate HFFz (Hydroxylfluorofurimazine) 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 substrate concentrate is mixed with the NanoLuc luciferase detection buffer in a volume ratio of 1:50 to detect the NanoLuc luciferase signal.

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

[0030] The AkaLuc luciferase detection buffer contains PBS buffer, sodium thiosulfate, and KSCN, which can effectively shield the fluorescence signal of NanoLuc luciferase with an inhibition rate of 99%, avoiding the influence of the fluorescence signal of NanoLuc luciferase on subsequent AkaLuc luciferase detection. After being prepared with AkaLuc luciferase substrate concentrate into AkaLuc luciferase detection working solution, the activity of AkaLuc luciferase can be detected without affecting the activity of AkaLuc luciferase.

[0031] Furthermore, the PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, and 1.84 mM KH2PO4.

[0032] Furthermore, the content of sodium thiosulfate in the AkaLuc luciferase detection buffer is 40-100 mM, preferably 60 mM.

[0033] Furthermore, the content of KSCN in the AkaLuc luciferase detection buffer is 20-50 mM, preferably 30 mM.

[0034] The AkaLuc luciferase substrate concentrate is Akalumine hydrochloride, and DMSO is used as a solvent to dissolve Akalumine hydrochloride to a content of 0.5-1 mg / mL, preferably 0.6 mg / mL. The AkaLuc luciferase substrate concentrate is mixed with the AkaLuc luciferase detection buffer at a volume ratio of 1:50 to detect the AkaLuc luciferase signal.

[0035] Furthermore, the preparation method of the substrate Akalumine hydrochloride is described in Chinese invention patent application CN117720477A.

[0036] In a second aspect, the present invention provides the use of the firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit as described in the first aspect in improving the detection sensitivity of the firefly luciferase reporter gene, the NanoLuc luciferase reporter gene and the AkaLuc luciferase reporter gene.

[0037] Firefly luciferase can catalyze luciferin to produce bioluminescent reaction. The reaction process is as follows:

[0038]

[0039] NanoLuc luciferase can catalyze the new substrate HFFz to produce a bioluminescent reaction. The reaction process is as follows:

[0040]

[0041] AkaLuc luciferase can catalyze the substrate Akalumine hydrochloride to produce a bioluminescent reaction. The reaction process is as follows:

[0042]

[0043] The present invention discloses a biological activity detection method, which comprises the following steps:

[0044] (1) Lysis of cells

[0045] (1.1) After aspirating the cell culture medium of cells transfected with firefly luciferase, NanoLuc luciferase, or AkaLuc 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. Pipette the required number of cells and collect the cells by centrifugation. After removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E7 cells / mL and incubate at room temperature for 5 minutes to fully lyse the cells.

[0046] (1.2) After sufficient lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant for determination.

[0047] (2) Firefly luciferase detection

[0048] (2.1) Take 50 μL of supernatant and add it to the 96-well plate to be tested. Add 50 μL of firefly luciferase detection reagent, shake the plate to mix, and detect the activity of firefly luciferase.

[0049] (3) NanoLuc luciferase assay

[0050] (3.1) According to the amount of 50 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and NanoLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare NanoLuc luciferase luciferase assay working solution;

[0051] (3.2) Add 50 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.

[0052] (4) AkaLuc luciferase assay

[0053] (4.1) According to the amount of 50 μL AkaLuc luciferase assay working solution required for each sample, take an appropriate amount of AkaLuc luciferase assay buffer and AkaLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare AkaLuc luciferase assay working solution;

[0054] (4.2) Add 50 μL of AkaLuc luciferase assay working solution to the 96-well plate to which NanoLuc luciferase assay working solution has been added in step (3.2), shake the plate to mix, and detect the activity of AkaLuc luciferase.

[0055] Compared with the prior art, the beneficial effects of this application are:

[0056] 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 luciferase detection without affecting the activity of NanoLuc luciferase. The AkaLuc luciferase detection buffer of the present invention can effectively shield the fluorescent signal of NanoLuc luciferase, with an inhibition rate of 99%, avoiding the influence of the fluorescent signal of NanoLuc luciferase on subsequent AkaLuc luciferase detection without affecting the activity of AkaLuc luciferase. The present invention detects firefly luciferase by using D-luciferin potassium salt as a substrate, introduces a new substrate HFFz (Hydroxylfluorofurimazine) for detecting NanoLuc luciferase luciferase, and introduces the independently synthesized Akalumine hydrochloride for detecting AkaLuc luciferase, so that the light signal intensity is large, the duration is long, and the sensitivity is high during the detection process, which is conducive to the high-throughput screening and use of three luciferases. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a graph showing the linear performance test results of the kit in Example 3 for detecting firefly luciferase.

[0058] Figure 2 This is a graph showing the linear performance test results of the kit in Example 3 for detecting NanoLuc luciferase.

[0059] Figure 3 This is a graph showing the linear performance test results of the kit in Example 3 for detecting AkaLuc luciferase.

[0060] Figure 4 This is a graph showing the signal stability detection results of firefly luciferase detected by the kit in Example 4.

[0061] Figure 5This is a graph showing the signal stability detection results of NanoLuc luciferase detected by the kit in Example 4.

[0062] Figure 6 This is a graph showing the signal stability detection results of AkaLuc luciferase detected by the kit in Example 4.

[0063] Figure 7 This is a comparison chart of the effects of different inhibitors on the inhibition rate of firefly luciferase in Example 5.

[0064] Figure 8 This is a bar graph of the signal detection results of NanoLuc luciferase in Example 7.

[0065] Fig. 9 This is a bar graph of the signal detection results of AkaLuc luciferase in Example 9. DETAILED DESCRIPTION

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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:

[0070] (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;

[0071] (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 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;

[0072] (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;

[0073] (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;

[0074] (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;

[0075] (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;

[0076] (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;

[0077] (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;

[0078] (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.

[0079] The preparation method of the Akalumine hydrochloride substrate involved in the present invention is described in Chinese invention patent application CN117720477A, ​​and the preparation steps are as follows:

[0080] (1) D-cysteine ​​methyl ester hydrochloride is dissolved in an acid solution, triphenylmethanol is added, the mixture is stirred at room temperature for reaction, and the mixture is concentrated under reduced pressure. After neutralization with an anion exchange resin, the mixture is washed with methanol, concentrated under reduced pressure, and purified to obtain intermediate 1;

[0081] (2) dissolving 4-dimethylaminocinnamaldehyde in a solvent, adding ethoxycarbonylmethylenetriphenylphosphine to react, drying under reduced pressure, extracting, and purifying to obtain intermediate 2;

[0082] (3) Intermediate 2 is dissolved in a solvent, and then an aqueous sodium hydroxide solution is added to heat and reflux, cooled to room temperature, and concentrated under reduced pressure to obtain a solid. The pH of the solid is adjusted to acidic, and the precipitated solid is filtered, rinsed, and dried to obtain Intermediate 3;

[0083] (4) Under nitrogen atmosphere, intermediate 3 and intermediate 1 were dissolved in a solvent, EDC solution was added, and then DMAP solution was added. The reaction was stirred at room temperature, extracted, and dried by spin drying. After purification, intermediate 4 was obtained;

[0084] (5) Under a nitrogen atmosphere, the intermediate 4 is dissolved in a solvent, triphenylphosphine oxide and trifluoromethanesulfonic anhydride are added to react, and then water is added to quench, extract, concentrate, and purify to obtain the intermediate 5;

[0085] (6) Under nitrogen atmosphere, the intermediate 5 is reacted with a solvent, extracted, and passed through a column to obtain Akalumine;

[0086] (7) Akalumine is sonicated in a solvent, allowed to stand after sonication, centrifuged, and the supernatant is collected and freeze-dried to obtain Akalumine hydrochloride.

[0087] 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.

[0088] Example 1

[0089] This embodiment provides a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit, which includes 6 reagents: luciferase lysate, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer, and AkaLuc luciferase substrate concentrate. The raw material composition and preparation method of each reagent are as follows.

[0090] (1) Preparation of luciferase lysate

[0091] 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.

[0092] 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.

[0093] 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.

[0094] (2) Preparation of firefly luciferase detection reagent

[0095] 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.

[0096] 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.

[0097] (3) Preparation of NanoLuc luciferase assay buffer

[0098] 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.

[0099] (4) Preparation of NanoLuc luciferase substrate (50×)

[0100] Measure 10 ml of DMSO and add it to the container. Weigh HFFz to make its concentration 0.65 mg / mL, which is the NanoLuc luciferase substrate concentrate.

[0101] (5) Preparation of AkaLuc luciferase assay buffer

[0102] 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 60 mM sodium thiosulfate and 30 mM KSCN. Finally, make the volume up to 50 ml with PBS buffer. The resulting transparent liquid is the AkaLuc luciferase detection buffer.

[0103] (6) Preparation of AkaLuc luciferase substrate concentrate

[0104] 10 ml of DMSO was measured and added to the container, and Akalumine hydrochloride was weighed to make its concentration 0.6 mg / mL, which is the AkaLuc luciferase substrate concentrate.

[0105] Example 2

[0106] This embodiment provides a firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit, which includes 6 reagents: luciferase lysate, firefly luciferase detection reagent, NanoLuc luciferase detection buffer, NanoLuc luciferase substrate concentrate, AkaLuc luciferase detection buffer, and AkaLuc luciferase substrate concentrate. The raw material composition and preparation method of each reagent are as follows.

[0107] (1) Preparation of luciferase lysate

[0108] In this embodiment, the luciferase lysate includes PBS buffer, non-ionic surfactant, metal ion shielding agent, reducing agent, glycerol, bovine serum albumin, trehalose and sucrose.

[0109] 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 the rest are glycerol, bovine serum albumin, trehalose, and sucrose.

[0110] 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, 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.

[0111] (2) Preparation of firefly luciferase detection reagent

[0112] 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.

[0113] 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.

[0114] (3) Preparation of NanoLuc luciferase assay buffer

[0115] 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.

[0116] (4) Preparation of NanoLuc luciferase substrate concentrate

[0117] 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 luciferase substrate concentrate.

[0118] (5) Preparation of AkaLuc luciferase assay buffer

[0119] 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 80 mM sodium thiosulfate and 45 mM KSCN. Finally, make the volume up to 50 ml with PBS buffer. The resulting transparent liquid is the AkaLuc luciferase detection buffer.

[0120] (6) Preparation of AkaLuc luciferase substrate concentrate

[0121] Measure 10 ml of DMSO and add it to the container, weigh Akalumine hydrochloride to make its concentration 1 mg / mL, which is the AkaLuc luciferase substrate concentrate.

[0122] Comparative Example 1:

[0123] 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 80 mM sodium thiosulfate. Finally, dilute to 50 ml with PBS buffer. The resulting transparent liquid is the AkaLuc luciferase detection buffer contrast reagent.

[0124] Comparative Example 2

[0125] 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 45 mM KSCN. Finally, dilute to 50 ml with PBS buffer. The resulting transparent liquid is the AkaLuc luciferase detection buffer contrast reagent.

[0126] Comparative Example 3

[0127] 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. Finally, dilute to 50 ml with PBS buffer. The resulting transparent liquid is the NanoLuc luciferase detection buffer contrast reagent.

[0128] Comparative Example 4

[0129] 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 12 mM EDTA. Finally, dilute to 50 ml with PBS buffer. The resulting transparent liquid is the NanoLuc luciferase detection buffer contrast reagent.

[0130] Comparative Example 5

[0131] 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.

[0132] Comparative Example 6

[0133] 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.

[0134] Example 3

[0135] The test kit linear performance test steps are as follows:

[0136] (1) Lysis of cells

[0137] (1.1) After aspirating the cell culture medium of cells transfected with firefly luciferase, NanoLuc luciferase, or AkaLuc 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. Pipette the required number of cells and collect the cells by centrifugation. After removing the supernatant, add luciferase lysis buffer to resuspend the cells to 1E7 cells / mL and incubate at room temperature for 5 minutes to fully lyse the cells.

[0138] (1.2) After sufficient lysis, centrifuge at 10,000-15,000×g for 3-5 minutes and take the supernatant as the test sample.

[0139] (2) The supernatant of step (1.2) was diluted with luciferase lysis buffer at a ratio of 1:2, 1:5, 1:10, 1:20, 1:50, and 1:100, respectively, to obtain a total of 7 samples to be tested.

[0140] (3) Firefly luciferase detection

[0141] (3.1) Take 50 μL of the supernatant of each sample in step (2) and add it to the 96-well plate to be tested, add 50 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 chemiluminescent signal of firefly luciferase.

[0142] (4) NanoLuc luciferase assay

[0143] (4.1) According to the amount of 50 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and NanoLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare NanoLuc luciferase assay working solution;

[0144] (4.2) Add 50 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 use a multi-function microplate reader (Berthokd LB941) to detect the chemiluminescent signal of NanoLuc luciferase.

[0145] (5) AkaLuc luciferase assay

[0146] (5.1) According to the amount of 50 μL AkaLuc luciferase assay working solution required for each sample, take an appropriate amount of AkaLuc luciferase assay buffer and AkaLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare AkaLuc luciferase assay working solution;

[0147] (5.2) Add 50 μL of AkaLuc luciferase detection working solution to the 96-well plate to which NanoLuc luciferase detection working solution has been added in step (4.2), shake the plate to mix, and use a multi-function microplate reader (Berthokd LB941) to detect the chemiluminescent signal of AkaLuc luciferase.

[0148] The above test was performed using the kits prepared in Example 1 and Example 2, and the test results were linearly fitted using the dilution ratio and the detection signal value (RLU) as curves.

[0149] The linearity test results of firefly luciferase are as follows Figure 1 The linearity test results of NanoLuc luciferase are shown in Figure 2 The linearity test results of AkaLuc luciferase are shown in Figure 3 As shown, it is shown that the linear fitting degree of the kit of the present invention is good and the linear range is wide.

[0150] Example 4

[0151] The test kit signal stability test, the test steps are as follows:

[0152] 1. Detection of Firefly Luciferase Signal Stability

[0153] The supernatant of step (1.2) of Example 3 was taken as the detection object, and 3 replicate wells were made. 50 μL of supernatant was taken from each well and added to a 96-well plate, and then 50 μL of firefly luciferase detection reagent was added. The plate was shaken to mix, and the chemiluminescent signal was detected using a multifunctional microplate reader (Berthokd LB941). The fluorescence value was set to be measured every 5 min, and the fluorescence value was measured for a total of 30 min. The average fluorescence values ​​at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min were read for subsequent analysis.

[0154] 2. Detection of NanoLuc luciferase signal stability

[0155] (1) According to the volume of 50 μL NanoLuc luciferase assay working solution required for each sample, take an appropriate amount of NanoLuc luciferase assay buffer and NanoLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare NanoLuc luciferase assay working solution;

[0156] (2) The supernatant of step (1.2) of Example 3 was taken as the detection object, and 3 replicate wells were made. 50 μL was taken from each well and added to a 96-well plate. 50 μL of NanoLuc luciferase detection working solution was added, and the plate was shaken to mix. The chemiluminescent signal was detected using a multifunctional microplate reader (Berthokd LB941). The fluorescence value was set to be measured every 5 minutes, and the fluorescence value was measured for a total of 30 minutes. The average fluorescence values ​​at 0 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes were read for subsequent analysis.

[0157] 3. Detection of AkaLuc luciferase signal stability

[0158] (1) According to the amount of 50 μL AkaLuc luciferase assay working solution required for each sample, take an appropriate amount of AkaLuc luciferase assay buffer and AkaLuc luciferase substrate concentrate at a volume ratio of 50:1 to prepare AkaLuc luciferase assay working solution;

[0159] (2) Take the supernatant of step (1.2) of Example 3 as the detection object, make 3 replicate wells, take 50 μL from each well and add it to a 96-well plate, add 50 μL AkaLuc luciferase detection working solution, shake the plate to mix, use a multifunctional microplate reader (BerthokdLB941) to detect the chemiluminescent signal, set the fluorescence value to be measured every 5 minutes, and measure the fluorescence value for a total of 30 minutes. Read the average fluorescence value at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min for subsequent analysis.

[0160] The above test was performed on Example 1 and Example 2.

[0161] The luminescence stability results of firefly luciferase detection are as follows Figure 4 The luminescence stability results of NanoLuc luciferase assay are shown in Figure 5 The luminescence stability results of AkaLuc luciferase assay are shown in Figure 6 As shown, it is explained that the detection signal of the kit of the present invention decreases slowly within 30 minutes and the signal is highly stable.

[0162] Example 5 Effects of different inhibitors on the inhibition rate of firefly luciferase

[0163] The experimental steps are as follows:

[0164] (1) Firefly luciferase assay

[0165] (1.1) Take 50 μL of the supernatant solution of step (1.2) of Example 3 and add it to a 96-well plate. Make three replicate wells, add 50 μL of firefly luciferase detection reagent, shake the plate to mix, use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal of firefly luciferase, and take the average value of the fluorescence signal for final analysis.

[0166] (2) NanoLuc luciferase assay

[0167] (2.1) In the 96-well plate to which the firefly luciferase detection reagent has been added in step (1.1), 49 μL of NanoLuc luciferase detection buffer is added to each well, the plate is shaken to mix, and the chemiluminescent signal is detected. The obtained signal value reflects the inhibitory effect of the buffer on the firefly luciferase catalytic reaction in the previous step.

[0168] (2.2) Add 1 μL of NanoLuc luciferase substrate concentrate to the 96-well plate to which NanoLuc luciferase detection buffer has been added in step (2.1), shake the plate to mix, and use a multi-function microplate reader (Berthokd LB941) to detect the activity of NanoLuc luciferase.

[0169] The above test was carried out using Example 1, Example 2 and Comparative Example 5 and Comparative Example 6.

[0170] 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 effects of conventional KI and resveratrol. It has no inhibitory effect on NanoLuc luciferase and does not affect the subsequent chemiluminescent signal determination, while KI and resveratrol will inhibit the activity of NanoLuc luciferase.

[0171] Example 6

[0172] The inhibitory effect of pyrophosphate and EDTA on firefly luciferase in NanoLuc luciferase assay buffer was tested. The test steps are as follows:

[0173] 1. Firefly Luciferase Assay

[0174] Take 50 μL of each of the 7 samples in step (2) of Example 3 and add them to a 96-well plate. Add 50 μL of firefly luciferase detection reagent to each of the 96-well plates to be tested, shake the plate to mix, and use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal of firefly luciferase.

[0175] 2. Inhibition rate detection

[0176] Add 49 μL of NanoLuc luciferase detection buffer to the 96-well plate in step 1, shake the plate to mix, and use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal. According to the formula inhibition rate = 1-(signal after adding NanoLuc luciferase detection buffer / firefly luciferase detection signal) × 100%, the inhibition rate measured for the 7 samples can be obtained.

[0177] The above test was performed on Example 1, Example 2, Comparative Example 3 and Comparative Example 4.

[0178] The results are shown in the table below. The NanoLuc luciferase detection buffer of Example 1 and Example 2 has a strong inhibitory effect on the fluorescence signal of firefly luciferase secreted by different numbers of cells, and the inhibition rate exceeds 99%. The inhibition rate of Example 3 is 76%-83%, indicating that pyrophosphate has a relatively good quenching effect on the fluorescence signal of firefly luciferase but does not meet the expected requirements. The inhibition rate of Example 4 is 25%-32%, indicating that EDTA, as a metal ion chelator, can chelate a certain amount of reactant magnesium ions to inhibit the catalytic reaction of firefly luciferase and weaken the fluorescence signal, but the inhibitory effect is weak. Adding pyrophosphate or EDTA alone to the system cannot meet the expected requirements. Only EDTA and pyrophosphate in the same system can quench the fluorescence signal of firefly luciferase to a great extent without affecting subsequent tests.

[0179]

[0180]

[0181] Example 7

[0182] Whether the inhibitors pyrophosphate and EDTA in the NanoLuc luciferase assay buffer affect the detection of NanoLuc luciferase, the test steps are as follows:

[0183] 1. Control group 1 test

[0184] The supernatant of step (1.2) of Example 3 was taken into a 96-well plate, and 3 replicate wells were set up. 49 μL of PBS buffer and 1 μL of NanoLuc luciferase substrate concentrate were added, and the plate was shaken to mix. The chemiluminescent signal was detected using a multifunctional microplate reader (Berthokd LB941), and the average fluorescence value was used for subsequent analysis.

[0185] 2. Experimental group test

[0186] The test method is as follows: take the supernatant of step (1.2) of Example 3 in a 96-well plate, set up 3 replicate wells, add 49 μL of NanoLuc luciferase detection buffer and 1 μL of NanoLuc luciferase substrate concentrate, shake the plate to mix, use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal, and perform subsequent analysis based on the average fluorescence value.

[0187] The NanoLuc luciferase detection buffer and NanoLuc luciferase substrate concentrate of Example 1 and Example 2 were used to perform the experimental group test according to the above test method. The test results are as follows: Figure 8As shown, there is no significant difference in the signals between Example 1 and Example 2 and the control group, and the detected fluorescence signals are close, indicating that the inhibitors pyrophosphate and EDTA do not affect the activity of NanoLuc luciferase.

[0188] Example 8

[0189] The inhibitory effect of sodium thiosulfate and KSCN on NanoLuc luciferase in AkaLuc luciferase assay buffer was tested. The test steps are as follows:

[0190] 1.NanoLuc luciferase assay

[0191] (1.1) Take 50 μL of each of the seven samples in step (2) of Example 3 and add them to a 96-well plate. According to the amount of 50 μL of NanoLuc luciferase assay working solution required for each sample, take appropriate amounts of NanoLuc luciferase assay buffer and NanoLuc luciferase substrate concentrate in a volume ratio of 50:1 to prepare NanoLuc luciferase assay working solution;

[0192] (1.2) Add 50 μL of NanoLuc luciferase detection working solution to each 96-well plate to be detected in step (1.1), shake the plate to mix, and use a multi-function microplate reader (Berthokd LB941) to detect the chemiluminescent signal of NanoLuc luciferase.

[0193] 2. Inhibition rate detection

[0194] Add 49 μL of AkaLuc luciferase detection buffer to the 96-well plate in step (1.2), shake the plate to mix, and use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal. According to the formula inhibition rate = 1-(signal after adding AkaLuc luciferase detection buffer / NanoLuc luciferase detection signal) × 100%, the inhibition rate measured for the 7 samples can be obtained.

[0195] The above test was performed on Example 1, Example 2, Comparative Example 1 and Comparative Example 2.

[0196] The results are shown in the table below. The AkaLuc luciferase detection buffers of Example 1 and Example 2 have a strong inhibitory effect on the fluorescence signal of NanoLuc luciferase secreted by different cell numbers, with an inhibition rate of more than 99%. The inhibition of NanoLuc luciferase in Comparative Example 1 and Comparative Example 2 increases as the number of cells decreases, but the inhibition rate is only 50-70%, indicating that only sodium thiosulfate or only KSCN in the buffer cannot achieve a good inhibitory effect on NanoLuc luciferase. Only when sodium thiosulfate and KSCN are present in the buffer at the same time can the fluorescence signal of NanoLuc luciferase be quenched to a great extent without affecting subsequent tests.

[0197]

[0198]

[0199] Example 9

[0200] The inhibitors sodium thiosulfate and KSCN in the AkaLuc luciferase assay buffer have any effect on the detection of AkaLuc luciferase. The test steps are as follows:

[0201] 1. Control group 2 test

[0202] The supernatant of step (1.2) of Example 3 was taken into a 96-well plate, and 3 replicate wells were set up. 49 μL of PBS buffer and 1 μL of AkaLuc luciferase substrate concentrate were added, and the plate was shaken to mix. The chemiluminescent signal was detected using a multifunctional microplate reader (Berthokd LB941), and the average fluorescence value was used for subsequent analysis.

[0203] 2. Experimental group test

[0204] The test method is as follows: take the supernatant of step (1.2) of Example 3 in a 96-well plate, set up 3 replicate wells, add 49 μL of AkaLuc luciferase detection buffer and 1 μL of AkaLuc luciferase substrate concentrate, shake the plate to mix, use a multifunctional microplate reader (Berthokd LB941) to detect the chemiluminescent signal, and perform subsequent analysis based on the average fluorescence value.

[0205] The AkaLuc luciferase detection buffer and AkaLuc luciferase substrate concentrate of Example 1 and Example 2 were used to perform experimental group tests according to the above test method. The test results are as follows: Fig. 9 As shown, there is no significant difference in the signals between Example 1 and Example 2 and the control group, and the detected fluorescence signals are close, indicating that the inhibitors sodium thiosulfate and KSCN do not affect the activity of AkaLuc luciferase.

[0206] 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 made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit, characterized in that: Includes Luciferase Lysate, Firefly Luciferase Assay Reagent, NanoLuciferase Assay Buffer, NanoLuciferase Substrate Concentrate, AkaLuc Luciferase Assay Buffer, and AkaLuc Luciferase Substrate Concentrate.

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, 1.84 mM KH2PO4; The content of pyrophosphate in the NanoLuc luciferase assay buffer is 30-50 mg / mL; The content of EDTA in the NanoLuc luciferase assay buffer is 8-12 mM.

8. The kit according to claim 1, characterized in that The NanoLuc luciferase substrate concentrate is HFFz, with a content of 0.6-0.8 mg / mL.

9. The kit according to claim 1, characterized in that The AkaLuc luciferase detection buffer contains PBS buffer, sodium thiosulfate, and KSCN.

10. The kit according to claim 9, characterized in that The PBS buffer contains 137.07 mM NaCl, 10 mM Na2HPO4, 1.84 mM KH2PO4; The content of sodium thiosulfate in the AkaLuc luciferase assay buffer is 40-100 mM; The content of KSCN in the AkaLuc luciferase assay buffer is 20-50 mM.

11. The kit according to claim 1, characterized in that AkaLuc luciferase substrate concentrate is Akalumine hydrochloride with a content of 0.5-1 mg / mL.

12. Use of the firefly-NanoLuc-AkaLuc triple luciferase reporter gene detection kit according to any one of claims 1 to 11 in improving the detection sensitivity of firefly luciferase reporter gene, NanoLuc luciferase reporter gene and AkaLuc luciferase reporter gene.

Images