Method for high-level expression of sweet taste receptor protein and application

Abstract

The invention belongs to the technical field of biological cells, and particularly relates to a method for high-level expression of sweet receptor protein and application. The method comprises the following steps: carrying out cell transformation and expanding culture on recombinant plasmids for expressing sweet taste receptors (T1R2 and T1R3); the recombinant plasmid is introduced into the HEK293 cell in a liposome transfection mode; t1R2 and T1R3 are jointly expressed to form a taste receptor of a corresponding sweet stimulant for sugar response test. According to the technical scheme, the exogenous DNA is directly introduced into cytoplasm or cell nucleus under specific conditions, and efficient and rapid expression of T1R2 and T1R3 sweet taste receptors in an in-vitro cell system can be realized in a short time without being integrated into a host genome. Meanwhile, operation is easy, convenient and rapid, complex and tedious operation steps are not needed, and time and labor cost can be saved; moreover, no gene integration risk exists, and potential side effects and uncertainty caused by gene editing are avoided.

Description

Technical Field

[0001] The invention belongs to the field of biological cell technology, and specifically relates to a method for expressing sweet taste receptor protein at a high level and application thereof. Background Art

[0002] Sweet taste receptors are heterodimers composed of two subunits, T1R2 and T1R3, and belong to the class C G protein-coupled receptors (GPCRs). In the taste system, T1R2 / T1R3 heterodimers are key receptors for human sweet taste recognition, and play an important role in the recognition of sweet molecules and the process of sweet signal transduction. However, the natural expression of these receptors in vitro is relatively low, which poses a major challenge to researchers in the high-throughput screening of sweet substances, functional properties, and sweetness mechanism research.

[0003] At present, the traditional stable transfection technology is the most commonly used method for researchers to obtain target proteins. This method integrates the target gene into the cell chromosome to achieve persistent expression of the target protein. Although this method can solve the above problems to a certain extent, the process is cumbersome, time-consuming, and has low protein expression. In addition, resistance screening and multiple passages of cells may lead to problems such as instability of the target genome and integration site effects, which increases the difficulty and uncertainty of the research. Therefore, there is an urgent need to develop a new transfection method to achieve efficient, rapid, and high-level expression of T1R2 and T1R3 sweet taste receptor proteins in in vitro cell systems. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of highly expressing sweet taste receptor proteins (T1R2, T1R3) by transient cell transfection, by which a large amount of highly expressed sweet taste receptor proteins can be obtained quickly and efficiently, and the protein can effectively respond to external sweet substances, thereby reflecting the sweetness of sweet compounds at a physiological level.

[0005] To achieve the above objectives, this application is implemented through the following technical solutions:

[0006] A method for highly expressing a sweet taste receptor protein, comprising the following steps:

[0007] S1. Treat the inner surface of the 6-well plate with Cell Adherent Reagent solution to form a cell adhesion coating;

[0008] S2. One day before transfection, take well-grown HEK-293 cells and inoculate them into the 6-well plate containing the cell adhesion coating in step S1. 2 , in a cell culture incubator at 37°C overnight;

[0009] S3. 3 h before transfection, the cultured HEK-293 cells were taken out from the above 6-well plate and observed under an inverted microscope. If the fusion rate reached the set value, transfection was performed. If the fusion rate did not reach the set value, transfection was not performed.

[0010] S4. Add the plasmid and P3000 auxiliary transfection reagent to opti-men to prepare liquid A, add lipo 3000 transfection reagent to opti-men to prepare liquid B, mix liquid A and liquid B respectively, and let stand at room temperature for 5 minutes;

[0011] S5. Add solution A to solution B to prepare transfection solution;

[0012] S6, discard the supernatant in the 6-well plate in step S3, add fresh culture medium, and evenly add the transfection solution prepared in step S5 to the 6-well plate;

[0013] S7. Place the 6-well plate with the transfection solution in a 5% CO 2 After incubating in a cell culture incubator at 37°C for 4-6 h, the transfection solution was discarded and the cell transfection was completed.

[0014] Furthermore, in step S1, the Cell Adherent Reagent solution and the PBS buffer solution were mixed evenly at a ratio of 1:200, and evenly coated in each well of a 6-well plate at room temperature. After 30 minutes, the excess solution was discarded to form a cell adhesion coating on the inner surface of each well of the 6-well plate.

[0015] Furthermore, in step S2, the inoculation volume of HEK-293 cells per well was 5 × 10 6 indivual.

[0016] Furthermore, in step S3, the set value of the fusion rate is 80%-90%.

[0017] Furthermore, in step S4, the plasmids are T1R2 and T1R3, and T1R2:T1R3=1:1.

[0018] Furthermore, the method further includes step S8, adding fresh complete culture medium to the 6-well plate in step S7 and continuing to culture for 48 hours to increase the number of transfected cells.

[0019] An application is the use of the highly expressed sweet taste receptor protein obtained by any of the above methods in response to sweet substances.

[0020] Furthermore, the change rate of cell fluorescence intensity is used to characterize the intensity of the cells' response to sweet substances.

[0021] Furthermore, the method of using the cell fluorescence intensity change rate to characterize the cell's response intensity to the sweet substance adopts the following steps:

[0022] Step 1: Prepare cell suspension and count with a hemocytometer. 4 The concentration of each well was transferred to a 96-well plate and placed in a 37°C, 5% CO 2 Incubate overnight in an incubator;

[0023] Step 2, take out the 96-well cell culture plate, discard the culture medium, and slowly add PBS to rinse twice;

[0024] Step 3, add 100 μL of 5 μmol / L fluorescent probe Fluo-4 AM working solution to each well and incubate at 37 °C for 40 min in the dark;

[0025] Step 4: After the incubation, remove the Fluo-4 AM working solution and wash the cells three times with PBS solution;

[0026] Step 5. Add 100 μL of HBSS solution to each well and incubate at 37 °C for 20 min in the dark.

[0027] Step 6, using the kinetic mode of the microplate reader to continuously detect the fluorescence intensity of the cells after adding the sweet substance several times;

[0028] Step 7: Use the rate of change of intracellular fluorescence intensity to reflect the response level of the cells to sweet substances.

[0029] Furthermore, the calculation formula for the change rate of intracellular fluorescence intensity is:

[0030] , where F max F is the fluorescence value with the strongest fluorescence intensity in the cell. 0 It is the weakest fluorescence value in the cell during the first 40 seconds.

[0031] The beneficial effects of the present invention are:

[0032] This technical solution is based on the direct introduction of exogenous DNA into the cytoplasm or nucleus under specific conditions, and can achieve efficient and rapid expression of T1R2 and T1R3 sweet taste receptors in an in vitro cell system in a short time without integration into the host genome. At the same time, the operation is simple and fast, without complicated and tedious operation steps, which can save time and labor costs; and there is no risk of gene integration, avoiding potential side effects and uncertainties caused by gene editing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing changes in intracellular fluorescence intensity of HEK-293 cells stimulated by sucrose solution (150 mM) according to an embodiment of the present invention;

[0034] Figure 2 A graph showing the rate of change of intracellular fluorescence intensity of HEK-293 cells stimulated by sucrose solution (150 mM) according to an embodiment of the present invention;

[0035] Figure 3 1 is a difference diagram of the change rate of the intracellular relative fluorescence intensity of HEK-293 cells stimulated by sucrose solutions of different concentrations according to an embodiment of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is described in detail below through examples. It should be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to limit the scope of the present invention. The change or adjustment of the relative relationship thereof shall also be regarded as the scope of the present invention without substantially changing the technical content.

[0037] In order to better understand the above technical scheme, the exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0038] The reagent materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] Example 1

[0040] A method for highly expressing sweet taste receptor proteins (T1R2, T1R3) by transient cell transfection, comprising the following steps:

[0041] S1. Pretreatment of 6-well plate: Mix Cell Adherent Reagent solution and PBS buffer solution at a ratio of 1:200, and evenly apply it to each well of the 6-well plate at room temperature. Discard the excess solution after 30 minutes. A cell adhesion coating can be formed on the inner surface of the 6-well plate. Dry it and set it aside (the cell adhesion coating prevents cells from falling off, increases cell adhesion, and is beneficial to the enhancement of cell signals).

[0042] S2. One day before transfection, take well-growing HEK-293 cells and add 5 × 10 6Cells / well were seeded in 6-well plates containing the above-mentioned cell adhesion coating and incubated in 5% CO 2 , in a cell culture incubator at 37°C overnight.

[0043] S3. 3 h before transfection, take out the cells cultured in the above 6-well plate and observe them under an inverted microscope. If the fusion rate (i.e., cell growth reaches 80%-90%), the transfection experiment can be carried out.

[0044] S4. Add 5 μg (T1R2: T1R3 = 1:1) plasmid and 5 μL of P3000 auxiliary transfection reagent to 150 μL of opti-men to prepare liquid A (P3000 auxiliary transfection reagent can increase the transfection efficiency of plasmids in cells), and add 5 μL of lipo3000 transfection reagent to 150 μL of opti-men to prepare liquid B. Gently mix liquid A and liquid B respectively, and let stand at room temperature for 5 minutes.

[0045] S5. Add solution A to solution B to prepare transfection solution. Mix by gently pipetting. Let stand at room temperature for 15 min before use.

[0046] S6. Aspirate and discard the supernatant of the 6-well plate in step S3, add fresh culture medium, and evenly add the entire transfection solution prepared in step S5 to the prepared 6-well plate.

[0047] S7. Place the 6-well plate containing the transfection solution in a 5% CO 2 After incubating in a cell culture incubator at 37°C for 4-6 h, the transfection solution was discarded and the cell transfection was completed.

[0048] S8. Add fresh complete culture medium to the 6-well plate in step S7 and continue culturing for 48 h to increase the number of transfected cells and the expression level of sweet taste receptor protein.

[0049] Example 2

[0050] This experiment measures the changes in intracellular calcium ion concentration under sweet substances based on fluorescence intensity, so as to further reflect the response of cells to sweet substances. The experimental steps are as follows:

[0051] Step 1: Prepare cell suspension and count with a hemocytometer. 4 The concentration of each well was transferred to a 96-well plate and placed in a 37°C, 5% CO 2 Incubate overnight in an incubator.

[0052] Step 2: Take out the 96-well cell culture plate, discard the culture medium, and slowly add PBS to rinse twice.

[0053] Step 3: Add 100 μL of fluorescent probe Fluo-4 AM working solution (5 μmol / L) to each well and incubate at 37 °C for 40 min in the dark.

[0054] Step 4: After the incubation, remove the Fluo-4 AM working solution and wash the cells three times with PBS solution.

[0055] Step 5. Add 100 μL of HBSS solution to each well and incubate at 37 °C for 20 min in the dark to ensure that the AM bodies are completely deesterified in the cells, increase the free Fluo-4 bodies in the cells, and improve the fluorescent probe labeling efficiency.

[0056] Step 6. Use the kinetic mode of the microplate reader to continuously detect the fluorescence intensity of the cells after adding sweet substances for several times (120 s), and designate the wells with the shortest kinetic detection interval as the designated wells for detection (optimize the fluorescence detection wavelength to increase the sensitivity of cell response: before optimization, the excitation wavelength is 494 nm and the emission wavelength is 516 nm; after optimization, the excitation wavelength is 495 nm and the emission wavelength is 518 nm).

[0057] HEK-293 cells were stimulated with sweet substances at different concentrations, and the fluorescence intensity after adding sweet substances at different concentrations was detected using the kinetic mode of an enzyme reader. The fluorescence intensity value was used as the vertical axis to plot a curve of the fluorescence intensity change over time.

[0058] Step 7: The intracellular fluorescence intensity change rate is used to reflect the cell's response level to sugar substances (this method can avoid the background effect caused by the residual fluorescent dye and truly reflect the calcium fluctuation of the receptor in response to the sugar ligand). The calculation formula for the intracellular fluorescence intensity change ratio is as follows:

[0059] , where F max F is the fluorescence value with the strongest fluorescence intensity in the cell. 0 It is the weakest fluorescence value in the cell during the first 40 seconds.

[0060] With fluorescence intensity as the ordinate, plot the relative fluorescence intensity change curve of cells under the stimulation of 150 mM sucrose solution ( Figure 1 ), which can be used to determine the intracellular Ca 2+ The change of concentration. Figure 1 It can be seen that after the experimental group HEK-293 (T1R2 / T1R3) cells were stimulated with 150 mM sucrose concentration, the intracellular fluorescence intensity value first increased and then decreased with the increase of time. After the control group cells (HEK-293) were stimulated with 150 mM sucrose concentration, the intracellular fluorescence intensity value had a certain trend of increasing fluorescence intensity within the first 40 s, and the fluorescence intensity of the cells tended to be stable with the passage of stimulation time.

[0061] In order to further accurately compare the difference in the response degree of the experimental group cells and the control group cells to sucrose molecules under the stimulation of 150 mM sucrose concentration, the ratio of the intracellular fluorescence intensity change (△F / F0) of the two groups of cells was calculated. The results are shown in Figure 2 As shown in Figure 2. Under the stimulation of 150 mM sucrose concentration, the change degree of intracellular calcium ions in the experimental group cells was significantly higher than that in the control group cells (p < 0.001).

[0062] Example 3

[0063] The changes in intracellular calcium ion concentrations in sucrose solutions of different concentrations were measured based on fluorescence intensity, and the experimental steps were as shown in Example 2.

[0064] In order to more intuitively show whether the response of the sweet taste receptor cell line HEK-293 (T1R2 / T1R3) to sucrose molecules is related to the concentration of sucrose, the ratio of the change in intracellular fluorescence intensity (△F / F0) of the two groups of cells stimulated by sucrose solutions of different concentrations was calculated, as shown in Figure 2. Figure 3 As shown in the figure, with the increase of sucrose concentration, the degree of change of intracellular fluorescence intensity in the experimental group showed an increasing relationship. This result shows that the response degree of sweet taste receptor cell line HEK-293 (T1R2 / T1R3) to sucrose is positively correlated with the concentration of sucrose. Further T value test was performed on the change rate of intracellular fluorescence intensity of the two groups of cells stimulated by sucrose solution of different concentrations. The results showed that the change of intracellular fluorescence intensity in the experimental group was significantly higher than that in the control group (p < 0.001). This result shows that the cells in the experimental group are more sensitive to the stimulation of sweet substances and the experimental design is effective.

[0065] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0066] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for highly expressing a sweet taste receptor protein, characterized in that: Use the following steps: S1. Treat the inner surface of the 6-well plate with Cell Adherent Reagent solution to form a cell adhesion coating; S2. One day before transfection, take well-grown HEK-293 cells and inoculate them into the 6-well plate containing the cell adhesion coating in step S1, and incubate them in a cell culture incubator with 5% CO2 and 37°C overnight; S3. 3 h before transfection, the cultured HEK-293 cells were taken out from the above 6-well plate and observed under an inverted microscope. If the fusion rate reached the set value, transfection was performed. If the fusion rate did not reach the set value, transfection was not performed. S4. Add the plasmid and P3000 auxiliary transfection reagent to opti-men to prepare liquid A, add lipo 3000 transfection reagent to opti-men to prepare liquid B, mix liquid A and liquid B respectively, and let stand at room temperature for 5 minutes; S5. Add solution A to solution B to prepare transfection solution; S6, discard the supernatant in the 6-well plate in step S3, add fresh culture medium, and evenly add the transfection solution prepared in step S5 to the 6-well plate; S7. Incubate the 6-well plate with transfection solution in a cell culture incubator at 5% CO2 and 37°C for 4-6 hours, then discard the transfection solution and the cell transfection is complete.

2. The method for highly expressing sweet taste receptor protein according to claim 1, characterized in that: In step S1, the Cell Adherent Reagent solution and the PBS buffer solution are mixed evenly at a ratio of 1:200, and evenly coated in each well of a 6-well plate at room temperature. After 30 minutes, the excess solution is discarded to form a cell adhesion coating on the inner surface of each well of the 6-well plate.

3. The method for highly expressing sweet taste receptor protein according to claim 1, characterized in that: In step S2, the inoculation volume of HEK-293 cells per well was 5 × 10 6 indivual.

4. The method for highly expressing sweet taste receptor protein according to claim 1, characterized in that: In step S3, the set value of the fusion rate is 80%-90%.

5. The method for highly expressing sweet taste receptor protein according to claim 1, characterized in that: In step S4, the plasmids are T1R2 and T1R3, and the ratio of T1R2:T1R3=1:

1.

6. The method for highly expressing sweet taste receptor protein according to claim 1, characterized in that: The method further includes step S8, adding fresh complete culture medium to the 6-well plate in step S7 and continuing to culture for 48 hours to increase the number of transfected cells.

7. An application, characterized in that: Use of the highly expressed sweet taste receptor protein obtained by the method of any one of claims 1 to 6 in the response to sweet substances.

8. The use according to claim 7, characterized in that: The change rate of cell fluorescence intensity is used to characterize the response intensity of cells to sweet substances.

9. The use according to claim 8, characterized in that: The method for characterizing the intensity of the response of cells to sweet substances by the rate of change of cell fluorescence intensity adopts the following steps: Step 1: Prepare cell suspension and count with a hemocytometer. 4 The concentration of cells / well was transferred to a 96-well plate and cultured overnight in a 37°C, 5% CO2 incubator; Step 2, take out the 96-well cell culture plate, discard the culture medium, and slowly add PBS to rinse twice; Step 3, add 100 μL of 5 μmol / L fluorescent probe Fluo-4 AM working solution to each well and incubate at 37 °C for 40 min in the dark; Step 4: After the incubation, remove the Fluo-4 AM working solution and wash the cells three times with PBS solution; Step 5. Add 100 μL of HBSS solution to each well and incubate at 37 °C for 20 min in the dark. Step 6, using the kinetic mode of the microplate reader to continuously detect the fluorescence intensity of the cells after adding the sweet substance several times; Step 7: Use the rate of change of intracellular fluorescence intensity to reflect the response level of the cells to sweet substances.

10. The use according to claim 9, characterized in that: The calculation formula of the intracellular fluorescence intensity change rate is: , where F max F0 is the fluorescence value with the strongest fluorescence intensity in the cell, and F1 is the fluorescence value with the weakest fluorescence intensity in the cell in the first 40 seconds.

Images