Renilla luciferase reporter gene detection kit

Abstract

The invention relates to a bioluminescence detection technology and a kit. The invention particularly discloses a renilla luciferase reporter gene detection kit which comprises a buffer solution and a detection substrate, and the buffer solution comprises Tris, potassium iodide, sodium sulfite, TCEP, a reducing agent and a surfactant. The prepared renilla luciferase reporter gene detection reagent has the advantages of high luminous intensity, low background and long luminous duration. When the renilla luciferase detection reagent is used, extra operation such as removal of a culture medium is not needed, and the expression level of renilla luciferase in cells can be rapidly detected.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly to a Renilla luciferase reporter gene detection kit. Background Art

[0002] A reporter gene refers to a group of genes encoding proteins or enzymes that are easily detectable. By fusing the coding sequence and regulatory sequence of the reporter gene, or fusing it with other target genes, under appropriate activation or inhibition conditions, the reporter gene can be expressed under the control of the regulatory sequence, and the expression regulation of the target gene can be "reported" by detecting the expression product of the reporter gene. As a reporter gene, it generally needs to meet the following characteristics: the sequence has been cloned or fully determined; the transfected cells do not contain the expression product; its expression product is easy to detect. Currently, the commonly used reporter genes are mainly divided into the following types: luciferase, β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), secreted human placental alkaline phosphatase (SEAP), green fluorescent protein (GFP), etc.

[0003] Luciferase is a class of enzymes that can catalyze the oxidation of different substrates to emit light, and mammalian cells do not have endogenous luciferase. The most commonly used luciferases are Firefly luciferase isolated from a kind of firefly and Renilla luciferase isolated from Renilla reniformis. Firefly luciferase is a protein with a molecular weight of 61 kDa. In the presence of ATP, Mg 2+ , and oxygen, it can catalyze the oxidation of luciferin to oxyluciferin, and during this process, light with a wavelength of about 560 nm will be emitted. Renilla luciferase is a protein with a molecular weight of 36 kDa. In the presence of oxygen, it can catalyze the oxidation of coelenterazine to coelenteramide, emitting light with a wavelength of about 480 nm.

[0004] Due to the chemical structure characteristics of the Renilla luciferase substrate coelenterazine, it will slowly oxidize and generate self-luminescence in solution, which will cause a relatively high background signal for reporter gene detection, thereby reducing the detection sensitivity. In addition, the luminescence signal of conventional Renilla luciferase reporter gene detection reagents decays very quickly, requires real-time detection, and has low throughput. Summary of the Invention

[0005] In order to solve the problems of high background, short signal half-life, and low throughput, the present application proposes a Renilla luciferase reporter gene detection kit, achieving the purposes of high luminescence intensity, low background, long luminescence duration, and simple operation.

[0006] The present invention is achieved through the following technical solutions. A Renilla luciferase reporter gene detection kit includes a buffer and a detection substrate. Using Renilla luciferase (rluc) can catalyze coelenterazine to produce a bioluminescence reaction, and the reaction process is as follows:

[0007]

[0008] The kit of the present invention uses a lysis buffer, which has high luminescence intensity, low background, long luminescence duration, and simple operation, 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. The buffer includes Tris, potassium iodide, sodium sulfite, TCEP, a reducing agent, and a surfactant.

[0009] The pH value of the buffer is 6.7 - 7.4, preferably 6.7.

[0010] The content of Tris in the buffer is 100 mM.

[0011] The content of potassium iodide in the buffer is 25 mM.

[0012] The content of sodium sulfite in the buffer is 125 - 250 mM, preferably 200 mM, which plays a role in reducing the background signal.

[0013] The content of TCEP in the buffer is 10 mM.

[0014] The content of the reducing agent DTT in the buffer is 1.25 - 5 mM, preferably 2.5 mM, and the reducing agent acts as a stabilizer.

[0015] The surfactant includes one of Triton X-100 and NP-40, and the volume content is 1 - 2%, preferably 1%, which acts as a cell lysing agent.

[0016] The detection substrate is coelenterazine, and the content is 50 - 100 μM, preferably 50 μM.

[0017] When the detection substrate is present, Renilla luciferase catalyzes the oxidation of coelenterazine in the presence of oxygen to generate coelenteramide, and at the same time produces blue light. The fluorescence brightness is proportional to the amount of luciferase.

[0018] The detection method is the following steps:

[0019] (1) Cell seeding

[0020] (1.1) Medium preparation

[0021] (1.2) Preheat the culture medium and 1× DPBS at 37°C;

[0022] (1.3) Remove the old culture medium and add 1 ml of DPBS to rinse the cells;

[0023] (1.4) Add 1 ml of 0.25% trypsin, then add culture medium to resuspend and count the cells; Transfer the centrifuged cells; After removing the supernatant, resuspend the cells to 2E5 cells / ml;

[0024] (1.5) Dilute the pGL4.45[rluc-ISRE-Hygro] Vector with Opti-MEM medium to 10 ng / mL;

[0025] (1.6) Add 3 μL of FuGENE® HD to 100 μL of 10 ng / mL plasmid solution and mix well, then let it stand at room temperature for 15 minutes;

[0026] (1.7) Slowly add 100 μL of the above solution to 2 mL of cell suspension and mix well. Add 100 μL of cell suspension to each well of a 96-well plate and culture at 37°C for 18 h.

[0027] (2) Cell stimulation detection

[0028] (2.1) IFN-α stimulation

[0029] Dilute IFN-α to 0.11 ng / mL. After IFN-α is serially diluted three-fold, take 10 μL each and add to the cells, then incubate at 37°C for 16 h;

[0030] After the cells are plated and stimulated with IFN-α, they express Renilla luciferase. After adding the detection reagent, a chemical reaction occurs, and the signal can be detected.

[0031] (2.2) Detection of Renilla luciferase

[0032] First, equilibrate the detection substrate and buffer to room temperature, then centrifuge to concentrate the liquid to the bottom of the bottle. Mix the detection substrate and buffer at a volume ratio of 1:100 to obtain the detection reagent. Add an equal volume of the detection reagent to the test cell samples, that is, add 100 μL of the detection reagent to the cells in 100 μL of culture medium, then shake for 3 - 5 minutes, and finally detect the chemiluminescence signal value.

[0033] Preferably, use Envision to detect the chemiluminescence signal value.

[0034] Compared with the prior art, the beneficial effects of the present application are as follows: The kit of the present invention can improve the signal value, reduce the background signal, and increase the signal stability by adding components and adjusting the ratio between components. Description of the Drawings

[0035] Figure 1 Schematic diagram of the chemiluminescence signal value detected by the kit in Example 1;

[0036] Figure 2 Comparison chart of the sensitivity between the kit prepared in the present application and similar products;

[0037] Figure 3 Comparison chart of the influence of the content of sodium sulfite on the test background value;

[0038] Figure 4 Comparison chart of the influence of the content of the reducing agent DTT on the test signal value;

[0039] Figure 5 Diagram of the influence of the composition of the surfactant on the test signal value;

[0040] Figure 6 Diagram of the influence of the change in the pH value of the buffer solution on the stability of the test signal value. Detailed Description of the Embodiments

[0041] The present invention will be further described in detail below through examples. All raw materials used in the examples can be purchased commercially or prepared by conventional methods.

[0042] Example 1: The kit consists of a buffer solution and a detection substrate.

[0043] Components and contents of the buffer solution: The buffer solution includes Tris 100 mM (pH 6.7), potassium iodide 50 mM, sodium sulfite 200 mM, TCEP 10 mM, reducing agent DTT 2.5 mM, and the volume content of Triton X-100 is 1%. The composition of the detection substrate is: coelenterazine 50 μM.

[0044] The kit consists of a buffer solution and a detection substrate.

[0045] Components and contents of the buffer solution: The buffer solution includes Tris 100 mM (pH 7.0), potassium iodide 50 mM, sodium sulfite 125 mM, TCEP 10 mM, reducing agent DTT 5 mM, and the volume content of NP-40 is 1%. The composition of the detection substrate is: coelenterazine 100 μM.

[0046] Example 3: The kit consists of a buffer solution and a detection substrate.

[0047] Buffer components and contents: The buffer includes 100 mM Tris (pH 6.7), 50 mM potassium iodide, 250 mM sodium sulfite, 10 mM TCEP, 2.5 mM reducing agent DTT, and 2% (by volume) Triton X-100. The detection substrate composition is: 50 μM coelenterazine.

[0048] Test Example 1.

[0049] (1) Reaction raw material model.

[0050] Reagent Model Article number Experimental parameters 293 cell line ATCC CRL-1573 20,000 cells / well pGL4.45[rluc-ISRE-Hygro] Vector Constructed by Yaoke - 50 ng / well Opti-MEM I Reduced Serum Medium, no phenol red Gibco 11058-021 - <![CDATA[FuGENE ® HD]]> Promega E2311 - IFN-α Genscript Z03002 1000 ng / ml; 3 fold dilution; 6 points Culture medium: DMEM with 4.5 g / L glucose, L-glutamine, sodium pyruvate Corning 10-013-CV - Fetal bovine serum Gibco 10099-141 10% 96-well plate PerkinElmer 6005680 -

[0051] (2) Detection method

[0052] (2.1) Cell seeding

[0053] (2.1.1) Preparation of culture medium;

[0054] (2.1.2) Preheat the culture medium and 1× DPBS at 37°C;

[0055] (2.1.3) Remove the old culture medium and add 1 ml of DPBS to wash the cells;

[0056] (2.1.4) Add 1 ml of 0.25% trypsin, then add the culture medium to resuspend and count the cells; Transfer and centrifuge to obtain the cells; After removing the supernatant, resuspend the cells to 2E5 cells / ml;

[0057] (2.1.5) Dilute pGL4.45[rluc-ISRE-Hygro] Vector with Opti-MEM medium to 10 ng / mL;

[0058] (2.1.6) Add 3 μL of FuGENE ® HD to 100 μL of 10 ng / mL plasmid solution, mix well, and let stand at room temperature for 15 minutes;

[0059] (2.1.7) Slowly add 100 μL of the above solution to 2 mL of cell suspension, mix well, add 100 μL of cell suspension to each well of a 96-well plate, and culture at 37°C for 18 h.

[0060] (2.2) Cell stimulation detection

[0061] (2.2.1) IFN-α stimulation

[0062] Dilute IFN-α to 0.11 ng / mL. After IFN-α is serially diluted three-fold, take 10 μL each and add to the cells, and incubate at 37°C for 16 h.

[0063] (2.2.2)Detection of Renilla luciferase

[0064] First, equilibrate the detection substrate and buffer in the kit of Example 1 to room temperature, then centrifuge to concentrate the liquid at the bottom of the bottle. Add the detection substrate to the buffer in a volume ratio of 1:100 and mix well to obtain the detection reagent. Add an equal volume of the detection reagent to the test cell sample, that is, add 100 μL of the detection reagent to the cells in 100 μL of the medium, then shake for 3 - 5 minutes, and finally detect the chemiluminescence signal value, as Figure 1 shown.

[0065] Comparative Example 1.

[0066] Use a commercially available Renilla luciferase reporter gene detection kit of Model A (promega, CAT: E2710) and perform the test according to the procedure of Test Example 1.

[0067] The test results are as Figure 2 shown, indicating that the kit prepared in this application has a higher luminescence intensity compared with the conventional commercially available kit.

[0068] Comparative Example 2.

[0069] Compared with Example 1, the sodium sulfite content in the kit is 125 mM.

[0070] Comparative Example 3.

[0071] Compared with Example 1, the sodium sulfite content in the kit is 250 mM.

[0072] The test comparison results are as Figure 3 shown: indicating that the sodium sulfite in the kit of this application can reduce the background value and improve the sensitivity.

[0073] Comparative Example 4.

[0074] Compared with Example 1, the DTT content in the kit is 1.25 mM.

[0075] Comparative Example 5.

[0076] Compared with Example 1, the DTT content in the kit is 5 mM.

[0077] The test comparison results are as Figure 4 shown: indicating that the DTT in the kit of this application will affect the test signal value.

[0078] Comparative Example 6.

[0079] Compared with Example 1, the volume content of Triton X - 100 in the kit is 1.5%.

[0080] Comparative Example 7.

[0081] Compared with Example 1, the volume content of Triton X-100 in the kit is 2%.

[0082] Comparative Example 8

[0083] Compared with Example 1, the volume content of NP-40 in the kit is 1%.

[0084] The test comparison results are as Figure 5 shown: It shows that the components of the surfactant in the kit of the present application will affect the test signal value.

[0085] Comparative Example 9

[0086] Compared with Example 1, the pH value of the buffer solution in the kit is 7.0.

[0087] Comparative Example 10

[0088] Compared with Example 1, the pH value of the buffer solution in the kit is 7.4.

[0089] The test comparison results are as Figure 6 shown: It shows that the pH value of the buffer solution in the kit of the present application affects the signal stability.

[0090] As described above, when using the kit of the present invention to detect cells, by optimizing the ratio and pH value of each component in the buffer solution, the signal value can be increased, the background signal can be reduced, and the signal stability can be increased.

Claims

1. A Renilla luciferase reporter gene detection kit, characterized in that, The described kit includes a buffer and a detection substrate.

2. The Renilla luciferase reporter gene detection kit according to claim 1, wherein Tris, potassium iodide, sodium sulfite, TCEP, reducing agent, surfactant.

3. The Renilla luciferase reporter gene detection kit according to claim 2, characterized in that, The pH value of the buffer is 6.7 - 7.4, preferably 6.

7.

4. The Renilla luciferase reporter gene detection kit according to claim 2, wherein The content of the Tirs is 100 mM.

5. The Renilla luciferase reporter gene detection kit according to claim 2, wherein The content of the potassium iodide is 25 mM.

6. The Renilla luciferase reporter gene detection kit according to claim 2, wherein The content of the sodium sulfite is 125 - 250 mM, preferably 200 mM, which functions to reduce the background signal.

7. The Renilla luciferase reporter gene detection kit according to claim 2, characterized in that, The content of the TCEP is 10 mM.

8. The Renilla luciferase reporter gene detection kit according to claim 2, characterized in that The content of the reducing agent DTT is 1.25 - 5 mM, preferably 2.5 mM, and the reducing agent functions as a stabilizer.

9. The Renilla luciferase reporter gene detection kit according to claim 2, wherein The surfactant includes one of Triton X-100 and NP-40, with a volume content of 1 - 2%, preferably 1%, which functions as a cell lysing agent.

10. The Renilla luciferase reporter gene detection kit according to claim 1, characterized in that, The detection substrate is coelenterazine, with a content of 50 - 100 μM.

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