Biological activity detection kit based on luciferase reporter gene

Abstract

The invention provides a biological activity detection kit based on a luciferase reporter gene. Comprising a substrate buffer solution and a firefly luciferase substrate, the substrate buffer solution comprises a zwitterionic buffer agent, glycerol, magnesium salt, a sulfydryl reducing agent, a high-energy compound, a coenzyme, a nonionic surfactant, a sulfo-nucleobase compound, an antioxidant, a metal ion masking agent and polyamine; the substrate buffer solution and a firefly luciferase substrate form a luciferase reporter gene-based biological activity detection test box; the kit not only has the advantage that one-step sample adding operation is simple, convenient and rapid, but also is not easily influenced by mixed sample adding conditions, good in result repeatability and more accurate in detection, meanwhile, the kit disclosed by the invention is brighter in light signal output and higher in sensitivity, and the detection luminescence value is higher than that of a kit of a similar product. And for some experiment systems for trace expression of firefly luciferase, the requirement of detecting the luminescence value can be met.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and specifically discloses a biological activity detection kit based on luciferase reporter gene with significantly improved thermal stability. Background Art

[0002] The reporter gene method is a method of integrating the reporter gene into the biological system to be tested by molecular biological means, and then studying specific biological processes by detecting the signal generated by the reporter gene. Firefly luciferase is an enzyme selected as a common reporter gene. It can catalyze the oxidation of the substrate luciferin. During the oxidation process, the luciferin substrate emits biological fluorescence with a wavelength of about 560nm. The fluorescence signal value can be accurately measured by a fluorescence meter, and then the expression of firefly luciferase can be accurately quantified. In the luciferase-luciferin reaction process, oxygen is needed to oxidize the substrate, adenosine triphosphate is used to provide energy, and divalent metal ions are also required to participate in the reaction, and finally a detectable fluorescence signal is emitted.

[0003] The luciferase reporter gene system is currently commonly used in the verification experiments of the regulatory functions of various expression elements, such as promoter transcription activity, transcription factor binding site verification, microRNA binding site verification, 5'UTR and 3'UTR function verification, enhancer / silencer function verification, effector element function verification, etc. Through this gene expression regulation verification related experiment, it can be more deeply and widely applied to the development and production quality control of biological drugs such as antibody drugs, such as antibody-dependent cell-mediated cytotoxicity (ADCC) effect experiments, which can achieve large-scale, high-accuracy, and high-efficiency biological activity detection, and can be oriented to many aspects including scientific research and production, with broad application scenarios and application depth.

[0004] However, luminescent substrates (e.g., luciferin) decompose during storage, resulting in loss of the substrate before addition or use in a bioassay. This decomposition may be due to the instability of the luminescent substrate in solution over time in a temperature-dependent manner. This decomposition wastes the luminescent substrate and reduces the sensitivity and reproducibility of luminescent measurements obtained from bioassays using the decomposed luminescent substrate. In addition, the products produced by this decomposition typically inhibit the luminescent reaction, resulting in decreased and inconsistent light output performance.

[0005] Currently, there are many one-step firefly luciferase detection reagents on the market. The operation is relatively simple, but the luminescence signal is low, and the storage stability of the substrate buffer and the substrate after mixing is poor, which is not convenient for long-term storage.

[0006] Therefore, there is a need for a luciferase reporter gene detection reagent that can be stored at a temperature higher than the ambient temperature for a long time, can detect the fluorescent signal quickly, sensitively and accurately, and can maintain a stable fluorescent signal during long-term detection operations. Summary of the invention

[0007] In order to solve the problem that reporter gene kits are sensitive to temperature and that conventional kits cannot be used to detect signals when the signals are weak due to cell-specific reasons or drug effects, the present invention discloses a biological activity detection kit based on luciferase reporter gene with significantly improved thermal stability, which has the advantages of significant thermal stability, high sensitivity, easy detection and good reproducibility.

[0008] The present invention includes the following technical solutions:

[0009] A biological activity detection kit based on luciferase reporter gene, characterized in that it comprises a substrate buffer and a luciferase substrate;

[0010] The substrate buffer comprises a zwitterionic buffer, glycerol, magnesium salt, sulfhydryl reducing agent, high energy compound, coenzyme A, surfactant, thionucleobase compound, antioxidant, metal ion masking agent and polyamine;

[0011] The zwitterionic buffer is selected from any one of phosphate buffer solution, citrate, HEPES, MOPS, MES and Tris-HCl, and the concentration in the buffer is 0.001%-10%, preferably 1%;

[0012] The concentration of glycerol in the buffer is 0.001%-30%, preferably 5%;

[0013] The magnesium salt is selected from any one of magnesium sulfate and magnesium chloride, and the concentration in the buffer is 0.01mM-80mM, preferably 10mM;

[0014] The thiol reducing agent is selected from any one of dithiothreitol and TCEP, and the concentration in the buffer is 0.001mM-20mM, preferably 2mM;

[0015] The high-energy compound is ATP, and its concentration in the buffer is 0.001mM-20mM, preferably 1mM;

[0016] The concentration of coenzyme A in the buffer is 0.001mM-50mM, preferably 1mM;

[0017] The surfactant is selected from any one of Tween 20 and Triton 100, and the concentration in the buffer solution is 0.001%-10%, preferably 1%;

[0018] The thionucleobase compound is 6-AZA-2-thiouracil-5-carboxylic acid, and the concentration is 0.001mM-20mM, preferably 2mM;

[0019] The antioxidant is ergothioneine, and the concentration is 0.001mM-20mM, preferably 4mM;

[0020] The metal ion masking agent is selected from any one of ethylenediaminetetraacetic acid, 1,2-cyclohexanediaminetetraacetic acid, aminotriacetic acid, and ethylene glycol diethyl ether diaminetetraacetic acid, and the concentration in the buffer is 0.001mM-20mM, preferably 10mM;

[0021] The polyamine is selected from any one of triethylenediamine and p-phenylenediamine, and the concentration is 0.001%-10%, preferably 0.1%.

[0022] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the zwitterionic buffer is MES, and the concentration is 10mM-300mM, preferably 100mM.

[0023] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the magnesium salt is magnesium chloride, and its concentration in the buffer solution is 0.01mM-80mM, preferably 10mM.

[0024] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the thiol reducing agent is TCEP, and the concentration in the buffer solution is 0.001mM-20mM, preferably 2mM.

[0025] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the surfactant is Tween 20, and its concentration in the buffer solution is 0.001%-10%, preferably 1%.

[0026] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the metal ion masking agent is ethylene glycol diethyl ether diamine tetraacetic acid, and its concentration in the buffer solution is 0.001mM-20mM, preferably 10mM.

[0027] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the polyamine is triethylenediamine, and the concentration is 0.001%-10%, preferably 0.1%.

[0028] Furthermore, in the above-mentioned biological activity detection kit based on luciferase reporter gene, the luciferin is D-luciferin, halogenated luciferin or salt form, and the concentration of the luciferin when dissolved in buffer for detection is 0.01mM-50mM, preferably 6mM.

[0029] On the other hand, the present invention also discloses the use of the above kit in determining the amount of luciferase in a sample.

[0030] In some embodiments, the present invention provides a method for determining the amount of luciferase in a sample, wherein the sample may contain cells expressing luciferase, and the content of luciferase in the sample is determined by detecting the luminescence intensity. The sample containing cells expressing luciferase may be untreated cells or lysed cell extracts.

[0031] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0032] The substrate buffer used in the kit of the present invention has the characteristics of remarkable thermal stability, 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 signal, weak drug effect, and multi-cell interaction signal. The buffer includes zwitterionic buffer, glycerol, magnesium salt, sulfhydryl reducing agent, high-energy compound, coenzyme, nonionic surfactant, thionucleobase compound, antioxidant, metal ion masking agent, polyamine. In some embodiments, the substrate buffer includes thiothymine analogs, wherein the thionucleobase analog is ATCA, and the effect is to inhibit substrate decomposition. Compared with the composition not including ATCA, when stored at a temperature of about 0°C to about 35°C, the reaction mixture can prolong storage stability, and in some embodiments, the buffer includes an antioxidant. The antioxidant is ergothioneine, and the half-life of the luminescent signal is increased compared with the reaction assay without ergothioneine. The half-life of the luminescent signal can be increased by at least about 20%.

[0033] The present invention discloses a biological activity detection kit based on luciferase reporter gene, which is a kit for detecting firefly luciferase reporter gene in a one-step method. The detection principle of the present invention is as follows: Figure 1 As shown, firefly luciferase can catalyze luciferin to produce a biological fluorescence reaction; the present invention has developed the components and concentration of the substrate buffer so that the kit not only has the advantages of a simple and fast one-step sample addition operation, but is also not easily affected by mixed sample addition conditions, the result repeatability is good, and the detection is more accurate. At the same time, the light signal output of the kit of the present invention is brighter and has higher sensitivity. The detected luminescence value is higher than that of similar product kits. For certain experimental systems that express firefly luciferase in trace amounts, it can also meet the customer's demand for detected luminescence values, and is more suitable for applications in a variety of biological detections. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the detection principle of the present invention;

[0035] Figure 2Schematic diagram of the difference between homogeneous detection and inhomogeneous detection;

[0036] Figure 3 This is the effect of ATCA on storage stability;

[0037] Figure 4 This is a graph showing the effect of ergothioneine on luminescence brightness and signal half-life;

[0038] Figure 5 This is the effect of triethylenediamine on the luminescence brightness and signal half-life;

[0039] Figure 6 Figure 2 shows the parallelism test of intracellular reporter gene response. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0041] The invention provides a luciferase reporter gene detection kit with good storage stability. The thermal stability of the kit is significantly improved. When the kit is stored at room temperature for 24 hours, the luminescence intensity is maintained at more than 85%.

[0042] The invention provides a luciferase reporter gene detection kit with a long signal half-life, and the half-life is significantly extended by 30%.

[0043] This kit is a homogenous test. The test reagent does not need to be lysed separately, the culture medium is removed, or multiple pipetting operations are performed. The cells can be lysed quickly and the results can be detected after 5-10 minutes, which improves efficiency and saves time. The difference between homogenous and non-homogeneous tests can be seen. Figure 2 shown.

[0044] Example 1

[0045] Verification of the storage stability of the thionucleobase compound ATCA (6-AZA-2-thiouracil-5-carboxylic acid).

[0046] The effect of ATCA (2mM) in the detection substrate buffer and the detection effect of the detection reagent stored at room temperature after mixing the substrate was compared to illustrate its effect on the storage stability of the reagent. The substrate buffer includes 100mM MES, 10mM magnesium chloride, 30mM DTT, 10mM EGTA, 1mM ATP, 1mM coenzyme A, 1% Tween 20, and the substrate is 0.01mM D-luciferin; the substrate buffer containing or not containing ATCA (2mM) was mixed with the substrate reagent and the commercially available P brand reagent at room temperature (22°C) for 2, 4, 6, 8, 12, and 24h, and then added to a black opaque 96-well plate with Firefly luciferase expressed by 293T cells in a 1:1 ratio, and the signal reading was detected by an ELISA reader. See Figure 3 The presence of ATCA in the substrate buffer enhances the room temperature storage stability of the reagents after mixing the substrates.

[0047] Example 2

[0048] The effect of different concentrations of ergothioneine in the substrate buffer on the half-life of the reagent signal was compared. The substrate buffer includes 100mM MES, 20mM magnesium chloride, 20mM DTT, 10mM EGTA, 1mM ATP, 1mM coenzyme A, 1% Tween 20, 2mM ATCA, and the substrate is 6mM D-luciferin; the commercially available P brand and luciferase reporter gene detection reagents containing 0mM, 2mM, and 4mM ergothioneine were mixed with samples containing firefly luciferase, and 100μL of each reagent was added to a 96-well plate of 293T cells expressing firefly luciferase, with 100μL cells (20,000 cells / well) in each well, incubated for 10min, and the signal reading was detected with an enzyme reader. See Figure 4 , ergothioneine is proportional to the signal half-life within a certain concentration range, and the signal half-life of 4mM ergothioneine is extended by 30% compared with that without ergothioneine.

[0049] Example 3

[0050] Effect of triethylenediamine on luminescence brightness and signal half-life

[0051] The effect of the presence or absence of triethylenediamine in the substrate buffer on the half-life of the reagent signal was compared. The substrate buffer includes 100mM MES, 20mM magnesium chloride, 30mM DTT, 20mM EGTA, 500μM ATP, 1mM coenzyme A, 1% Tween 20, 2mM ATCA, 4mM ergothioneine, and the substrate is 50mM D-luciferin. The commercially available P brand and luciferase reporter gene detection reagents containing 0.01%, 0.05%, and 0.1% triethylenediamine were mixed with samples containing firefly luciferase. 100μL of each reagent was added to a 96-well plate of 293T cells expressing firefly luciferase, with 100μL cells (20,000 cells / well) in each well, incubated for 10 minutes, and the signal reading was detected using an ELISA reader. See Figure 5 , triethylenediamine prolongs the signal half-life of the reporter gene reagent.

[0052] Example 4

[0053] Parallelism testing of intracellular reporter gene responses.

[0054] The vector encoding firefly luciferase under the control of the promoter containing the NF-κB response element was transiently transfected into HEK293T cells. 24 hours after transfection, the luminescent signal was detected after TNF-α concentration gradient induction (6 hours). Control experiment: The experimental conditions were the same, and the commercially available P brand detection reagent was used as the control experiment. Five groups of parallel experiments were performed on the formula of this embodiment and the commercially available P brand detection reagent, and the CV value was calculated. The substrate buffer of this embodiment is: 120mM MES, 30mM magnesium chloride, 40mM DTT, 10mM EGTA, 2mM ATP, 2mM coenzyme A, 0.5% Tween 20, 2mM ATCA, 2mM ergothioneine, 0.1% triethylenediamine. The substrate is 6mM D-luciferin. See Figure 6 As shown in Table 1, the signal reading of Firefly in this embodiment is positively correlated with the drug concentration, and the change is more significant than that of brand P, which improves the signal value and signal window. In addition, the reagent in this embodiment has good reproducibility, with CV < 5%.

[0055] Table 1 Performance test

[0056]

[0057] It can be seen from the above examples that the present invention provides a luciferase reporter gene detection kit with good storage stability, whose thermal stability is significantly improved, and the luminescence intensity is maintained above 85% when stored at room temperature for 24 hours.

[0058] The invention provides a luciferase reporter gene detection kit with a long signal half-life, and the half-life is significantly extended by 30%.

[0059] This kit is a homogenous test. The detection reagent does not need to be separately lysed into cells, the culture medium is removed, or multiple pipetting operations are performed. The cells can be lysed quickly and the results can be detected after 5-10 minutes, which improves efficiency and saves time.

[0060] The reporter gene detection reagent developed by the present invention, including substrate buffer and matching substrate, has achieved remarkable progress in sensitivity, luminescence stability and storage stability, so that the overall performance of the reagent can meet both scientific research on gene expression regulation and enterprise new drug development. On the one hand, it can be applied to the field of scientific research, such as evaluating how response elements control gene expression; on the other hand, it can be applied to the evaluation of drug action mechanism, biological activity, efficacy and stability in new drug development and production.

[0061] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A biological activity detection kit based on luciferase reporter gene, characterized in that: Includes substrate buffer and luciferase substrate; The substrate buffer comprises a zwitterionic buffer, glycerol, magnesium salt, sulfhydryl reducing agent, high energy compound, coenzyme A, surfactant, thionucleobase compound, antioxidant, metal ion masking agent and polyamine; The zwitterionic buffer is selected from any one of phosphate buffer solution, citrate, HEPES, MOPS, MES and Tris-HCl, and the concentration in the buffer is 0.001%-10%, preferably 1%; The concentration of glycerol in the buffer is 0.001%-30%, preferably 5%; The magnesium salt is selected from any one of magnesium sulfate and magnesium chloride, and the concentration in the buffer is 0.01mM-80mM, preferably 10mM; The thiol reducing agent is selected from any one of dithiothreitol and TCEP, and the concentration in the buffer is 0.001mM-20mM, preferably 2mM; The high-energy compound is ATP, and its concentration in the buffer is 0.001mM-20mM, preferably 1mM; The concentration of coenzyme A in the buffer is 0.001mM-50mM, preferably 1mM; The surfactant is selected from any one of Tween 20 and Triton 100, and the concentration in the buffer solution is 0.001%-10%, preferably 1%; The thionucleobase compound is 6-AZA-2-thiouracil-5-carboxylic acid, and the concentration is 0.001mM-20mM, preferably 2mM; The antioxidant is ergothioneine, and the concentration is 0.001mM-20mM, preferably 4mM; The metal ion masking agent is selected from any one of ethylenediaminetetraacetic acid, 1,2-cyclohexanediaminetetraacetic acid, aminotriacetic acid, and ethylene glycol diethyl ether diaminetetraacetic acid, and the concentration in the buffer is 0.001mM-20mM, preferably 10mM; The polyamine is selected from any one of triethylenediamine and p-phenylenediamine, and the concentration is 0.001%-10%, preferably 0.1%.

2. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The zwitterionic buffer is MES, and the concentration is 10 mM-300 mM, preferably 100 mM.

3. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The magnesium salt is magnesium chloride, and its concentration in the buffer is 0.01 mM-80 mM, preferably 10 mM.

4. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The thiol reducing agent is TCEP, and the concentration in the buffer is 0.001 mM-20 mM, preferably 2 mM.

5. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The surfactant is Tween 20, and its concentration in the buffer is 0.001%-10%, preferably 1%.

6. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The metal ion masking agent is ethylene glycol diethyl ether diamine tetraacetic acid, and its concentration in the buffer is 0.001 mM-20 mM, preferably 10 mM.

7. A biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The polyamine is triethylenediamine, and the concentration is 0.001%-10%, preferably 0.1%.

8. The biological activity detection kit based on luciferase reporter gene according to claim 1, characterized in that: The fluorescein is D-fluorescein, halogenated fluorescein or a salt form. The concentration of the fluorescein when dissolved in a buffer for detection is 0.01 mM-50 mM, preferably 6 mM.

9. Use of the kit according to any one of claims 1 to 8 in determining the amount of luciferase in a sample.

10. The use according to claim 9, characterized in that: The samples contain cells expressing luciferase, and the cells are either untreated cells or lysed cell extracts.

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