Sweet taste sensing cell as well as preparation method and application thereof

Abstract

The invention discloses a sweetness sensing cell as well as a preparation method and application thereof, and belongs to the technical field of biology. The preparation method of the sweet taste sensing cell comprises the following steps: instantaneously transfecting a recombinant vector for expressing T1R2 and T1R3 into a eukaryotic cell; the mass ratio of the recombinant vector for expressing T1R2 to the recombinant vector for expressing T1R3 is 1: (1-2); the nucleotide sequence of the T1R2 is as shown in SEQ ID NO. 1; the nucleotide sequence of the T1R3 is as shown in SEQ ID NO. 2. The construction method of the sweetness sensing cell is provided by optimizing conditions such as T1R2 and T1R3 recombinant plasmid sequences, plasmid dosage, transfection reagent dosage, transfection time and the like, and the sweetness sensing cell with high sensitivity can be simply, conveniently and quickly prepared. The method can be matched with a flow cytometer, FLIPR or a microplate reader and the like for use, and is applied to rapid screening of sweet substances and quantitative analysis of sweetness values of the sweet substances.

Description

Technical Field

[0001] The invention relates to a sweet taste sensing cell and a preparation method and application thereof, belonging to the field of biotechnology. Background Art

[0002] Taste is the sensation produced when a substance is placed in the mouth, especially when specific receptors present on the surface of the tongue bind to the substance. The taste of mammals consists of five basic tastes, namely salty, sour, sweet, umami and bitter, and is considered to be formed by integrating these basic tastes. Among them, since sweetness can make humans and most mammals feel pleasant, sweetness has become one of the favorite tastes of humans and most mammals. At the same time, sweetness also indicates that the food contains nutrients, usually indicating more energy. Therefore, excessive intake of sweet substances can cause obesity and its related diseases, so in-depth research on the molecular mechanism of sweetness chemical perception has a very important impact and significance on the development of the food industry and the health of consumers. Actively studying and searching for sweeteners with good taste, harmless to the human body and low calories is closely related to human life and is of great significance to improving human living standards.

[0003] Sweet compounds are diverse and can be divided into natural sweeteners and artificial sweeteners. Natural sweeteners include certain natural sugars, sugar alcohols, proteins and amino acids; artificial sweeteners include some aminosulfonic acid lipids, dipeptides, halogenated sugars and sulfonylamides. These sweet compounds can activate the sweetness perception pathway by binding to sweet receptors, thereby producing sweetness. Therefore, sweet receptors are a key link in the perception of sweetness. Sweet receptors belong to the C-class GPCR family and form a heterodimeric protein complex (T1R2 / T1R3) with two subunits T1R2 and T1R3. The activated sweet receptors can regulate the intracellular calcium ion concentration through the GPCR-Gs-cNMP and GPCR-Gα / Gβγ-IP3 pathways, thereby changing the cell membrane potential and releasing neurotransmitters to transmit sweet information.

[0004] The strength of sweetness can be expressed by sweetness intensity, which is an important indicator of sweeteners. At present, the methods for detecting sweetness are mainly divided into two categories, namely analytical chemistry detection methods and biological detection methods. Among them, analytical chemistry detection methods mainly include high performance liquid chromatography, electronic tongue detection methods, sweetness meter detection methods, spectrophotometer detection methods, etc., which can accurately measure the content of sweet substances, but cannot quantitatively analyze the sweetness intensity at the physiological level. The biological method mainly relies on manual evaluation. Although this method can quantify sweetness at the physiological level, the subjectivity of manual evaluation is strong, and the accuracy of sweetness quantification is poor.

[0005] The Chinese invention patent application document with a publication date of January 4, 2019 and publication number CN109142298A disclosed a method for quantifying sweetness at the cellular level. By constructing a cell line that stably expresses T1R2 and T1R3 sweet taste receptors and using calcium ion imaging technology, it is possible to simulate and quantify the degree of perception of sweet taste receptors to sweet substances at the cellular level. This method can quickly, efficiently and sensitively detect the sweetness of compounds. However, this method requires a long period of drug screening experiments to obtain a cell line that stably expresses two sweet taste receptors at the same time, and there are problems such as long cell line preparation time, low cell viability, poor morphology, low expression of sweet taste receptors, disruption of normal cell metabolism and weak response to sweet substances. Therefore, the simple and rapid preparation of sweet taste sensing cells with high sensitivity has become a technical problem that needs to be solved urgently. Summary of the invention

[0006] The first object of the present invention is to provide a method for preparing sweet taste receptor cells to solve the problems of complex preparation, long cycle and poor sensitivity of sweet taste receptor cells in the prior art.

[0007] The second object of the present invention is to provide a sweet taste receptor cell to solve the problem of poor sensitivity of sweet taste receptor cells in the prior art.

[0008] The third object of the present invention is to provide an application of sweet taste receptor cells in the rapid screening of sweet substances and the quantitative analysis of the sweetness values ​​of sweet substances, so as to solve the problem of poor sensitivity of sweet taste receptor cells in the prior art.

[0009] In order to achieve the above object, the technical solution adopted in the method for preparing sweet taste sensing cells of the present invention is: A method for preparing sweet taste sensing cells comprises the following steps: transiently transfecting a recombinant vector expressing T1R2 and T1R3 into a eukaryotic cell; the mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 is 1:(1-2); the nucleotide sequence of T1R2 is shown in SEQ ID NO.1; the nucleotide sequence of T1R3 is shown in SEQ ID NO.2.

[0010] The beneficial effect of the above scheme is that the method for preparing sweet taste receptor cells of the present invention is an improved invention. The present invention has found through a large number of studies that the sweet taste receptors T1R2 and T1R3 have similar structures, and the N-termini of the two receptors have a Venus flytrap domain for identifying different sweet substances, while the sweet taste receptor sequences used in the prior art have added a histidine tag or a Flag tag to the N segment of the sweet taste receptor, which may affect the receptor's recognition and binding of sweet substances. The T1R2 and T1R3 sequences used in the present invention remove the histidine tag and the Flag tag of the N segment. The results show that the receptors after removing the tags can better identify sweet substances, effectively improving the sensitivity of the prepared sweet taste receptor cells.

[0011] Furthermore, experiments have shown that the sensitivity of eukaryotic cell lines stably expressing T1R2 and T1R3 is lower than that of eukaryotic cell lines transiently expressing T1R2 and T1R3. At the same time, the present invention optimizes the amount of vector used, and can quickly and easily prepare sweet taste sensing cells with higher sensitivity without the need for cumbersome cell screening processes, greatly simplifying the preparation process and cycle, and helping to save production costs in practical applications.

[0012] As a further improvement, the mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 is 1:(1.2-2).

[0013] The beneficial effect of the above scheme is that: in the prior art, the human sweet taste receptor is a heterodimeric protein complex composed of T1R2 and T1R3 (T1R2 / T1R3), and each receptor contains a T1R2 subunit and a T1R3 subunit. The present invention finds that when the mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 during transfection is 1: (1.2~2), the sweet taste receptor expression level in the constructed sweet taste sensing cells is high, and the cell morphology and normal growth are not affected, and the response to sweet substances is strong and the sensitivity is high.

[0014] Further preferably, the mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 is 1:(1.2-1.5).

[0015] As a further improvement, the transient transfection time is 24 to 36 h.

[0016] The beneficial effect of the above scheme is that the transfection time is within the above range, which ensures a high expression level of sweet taste receptors in sweet taste cells while not affecting the morphology and normal growth of the cells.

[0017] As a further improvement, the amount of transfection reagent used in the transient transfection is 4.5-5.5 μL.

[0018] The beneficial effect of the above scheme is that the amount of transfection reagent added during transfection is within the above range, which ensures a high expression level of sweet taste receptors in sweet taste cells while not affecting the morphology and normal growth of the cells.

[0019] As a further improvement, the preparation method includes transiently transfecting a recombinant vector expressing a calcium ion indicator protein and a recombinant vector expressing T1R2 and T1R3 into eukaryotic cells.

[0020] The beneficial effect of the above scheme is that: in the prior art, by constructing a cell line that stably expresses T1R2 and T1R3 sweet taste receptors, and using a calcium ion fluorescent probe to detect changes in intracellular calcium ions, it is possible to simulate and quantify the degree of human perception of sweet substances at the cellular level. However, the use of fluorescent probes for calcium ion detection has the following disadvantages: 1) The probe needs to be incubated before each detection, and the incubation efficiency of the probe is difficult to control, resulting in different fluorescence intensities produced by different batches of different cells, affecting the accuracy of the quantitative results; 2) The fluorescent probe has background fluorescence interference, resulting in a low signal-to-noise ratio of the imaging result; 3) The fluorescent probe is cytotoxic, which will affect cell viability and reduce the cell's response to sweet substance stimulation. The present invention constructs a cell line that can simultaneously express genetically encoded calcium ion indicators, T1R2 and T1R3, and can detect changes in intracellular calcium ions without the assistance of fluorescent probes. It can effectively solve the shortcomings of fluorescent probes in calcium ion detection, improve the sensitivity of detection, and greatly reduce the cost of the experiment.

[0021] As a further improvement, the genetically encoded calcium ion indicators include aequorin-based GAP series and calmodulin-based GCaMP series.

[0022] The beneficial effect of the above scheme is that: using the GAP series based on aequorin and the GCaMP series based on calmodulin as calcium ion indicator proteins, a cell line that can simultaneously express three proteins, namely, genetically encoded calcium ion indicators, T1R2 and T1R3, can be constructed, and changes in intracellular calcium ions can be detected without the assistance of fluorescent probes, which helps to improve the sensitivity of cells to sweet substances. In addition, the use of aequorin to detect calcium ion concentration avoids the use of calcium ion fluorescent probes, which can greatly reduce the cost of the experiment, eliminate the problem of unstable efficiency of fluorescent probe loading into cells, and reduce cytotoxicity.

[0023] As a further improvement, the mass ratio of the recombinant vector expressing the gene encoding the calcium ion indicator, the recombinant vector expressing T1R2, and the recombinant vector expressing T1R3 is (2-3):1:(1.2-2).

[0024] As a further improvement, the eukaryotic cells include HEK293 cells.

[0025] In order to achieve the above-mentioned object, the technical scheme adopted by the sweet taste receptor cells prepared by a method for preparing sweet taste receptor cells in the present invention is: A method for preparing sweet taste receptor cells. The sweet taste receptor cells are prepared.

[0026] The beneficial effect of the above scheme is that the sweet taste sensing cells of the present invention have a high sensitivity to sweet taste while maintaining the original morphology and vitality of the cells, so that most cells can respond to the stimulation of sweet substances, effectively improving the response of sweet substances.

[0027] In order to achieve the above-mentioned object, the technical solution adopted by the application of a sweet taste receptor cell in the rapid screening of sweet substances and the quantitative analysis of the sweetness value of sweet substances in the present invention is: A sweet taste receptor cell is used in the rapid screening of sweet substances and the quantitative analysis of the sweetness value of sweet substances.

[0028] The beneficial effect of the above scheme is that the morphology and vitality of the sweet taste sensing cells constructed by the present invention are not greatly affected, and experiments have proved that they can better identify sweet substances, effectively improve the sensitivity of sweet taste cells, and are conducive to improving the sensitivity of rapid screening of sweet substances and quantitative analysis of the sweetness values ​​of sweet substances.

[0029] Furthermore, sweet taste-receiving cells that co-express genes encoding three proteins, namely, calcium ion indicator, T1R2 and T1R3, can detect changes in intracellular calcium ions without the assistance of fluorescent probes, thus avoiding the use of calcium ion fluorescent probes, eliminating the problem of unstable efficiency of fluorescent probe loading into cells, reducing cytotoxicity, and greatly reducing the cost of the experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the sequence of the recombinant vector expressing T1R2 in Example 1 of the present invention; Figure 2 Schematic diagram of the sequence of the recombinant vector expressing T1R3 in Example 1 of the present invention; Figure 3 This is a graph showing the response of cells to sweet substances before and after sequence optimization in Example 1 of the present invention (20×); Figure 4 The effect of the amount of plasmid used in Example 2 of the present invention on the response of cells to sweet substances (20×); Figure 5 The effect of the amount of transfection reagent on the response of cells to sweet substances in Example 2 of the present invention (20×); Figure 6 The effect of transfection time on the response of cells to sweet substances in Example 2 of the present invention (20×); Figure 7 This is the immunoblotting test result of the sweet taste receptor cell 1 in Example 3 of the present invention; Figure 8 The results of fluorescence quantitative PCR detection of sweet taste receptor cells 1 in Example 3 of the present invention; Fig. 9 A comparison diagram of the responses of the stably transfected cells and the sweet taste sensing cells 1 to the cellular sweet substances in Example 3 of the present invention (20×); Fig.10 The effect of the amount of plasmid used on the response of cells to sweet substances in Example 4 of the present invention (20×); Fig.11 This is the response of the sweet taste receptor cell 2 to the cellular sweet substance in Example 4 of the present invention (20×). DETAILED DESCRIPTION

[0031] In the prior art, a long period of drug screening tests are required to obtain sweet taste sensing cells that stably express both T1R2 and T1R3 sweet taste receptors, and there are problems such as low cell viability, poor morphology, low sweet taste receptor expression, weak response to sweet substances, and disruption of normal cell metabolism. Based on this, the present invention provides a method for constructing sweet taste sensing cells by optimizing the T1R2 and T1R3 recombinant plasmid sequences, plasmid usage, transfection reagent usage, transfection time and other conditions, which can easily and quickly prepare highly sensitive sweet taste sensing cells. It can be used in conjunction with instruments such as flow cytometers, FLIPR or microplate readers, and can be applied to the rapid screening of sweet substances and the quantitative analysis of the sweetness values ​​of sweet substances.

[0032] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0033] An embodiment of a sweet taste sensing cell and a preparation method and application thereof in the present invention: Example 1 Construction of recombinant vector expressing T1R2 or T1R3 and cell transfection In this example, HEK-293 cells were used as templates to obtain T1R2 and T1R3 gene fragments, which were inserted into plasmid vectors to construct recombinant expression vectors expressing T1R2 and T1R3, respectively. The specific implementation methods are as follows: 1. Construction of recombinant vector Total RNA was extracted from HEK-293 cells and reverse transcribed to obtain cDNA. T1R2 and T1R3 specific primers were used for PCR amplification to obtain T1R2 and T1R3 gene fragments (the nucleotide sequence of T1R2 is shown in SEQ ID NO.1, and the nucleotide sequence of T1R3 is shown in SEQ ID NO.2). The gene fragments were inserted into plasmid vectors (the T1R2 gene fragment was inserted into the pcDNA3.1 plasmid vector and the T1R3 gene fragment was inserted into the pEGFP plasmid vector) by double restriction enzyme digestion (the restriction sites were BamH I and Xho I, respectively). After transformation and single clone screening, T1R2 and T1R3 recombinant vectors were obtained. Finally, the recombinant plasmid sequence was verified by Sanger sequencing, and the verified recombinant plasmids were stored at -80°C.

[0034] Among them, the specific primer sequence of T1R2 is as follows: T1R2-F: 5'-TGGGAGGTCTATATAAGCAGAG-3' (as shown in SEQ ID NO. 3); T1R2-R: 5′-ACAGTGGGAGTGGCACCTTC-3′ (as shown in SEQ ID NO. 4).

[0035] The specific primer sequences for T1R3 are as follows: T1R3-F: 5'-AGCGTCCTCCTGTTCCCTGG-3' (as shown in SEQ ID NO. 5); T1R3-R: 5'-AAACCTCTACAAATGTGGTATG-3' (as shown in SEQ ID NO. 6).

[0036] The sequence diagram of the recombinant vector expressing T1R2 (hereinafter referred to as T1R2 recombinant vector) is as follows Figure 1 The sequence diagram of the T1R3 recombinant vector (hereinafter referred to as the T1R3 recombinant vector) is shown in Figure 2 As shown. Figure 1 and Figure 2 At the same time as the recombinant vectors, a control recombinant vector was also constructed. The control vector of the T1R2 recombinant vector added a six-histidine tag at the N-terminus of the T1R2 sequence, and the control vector of the T1R3 recombinant vector added a Flag tag at the N-terminus of the T1R3 sequence. The rest of the structures were the same.

[0037] 2. Optimization of vector sequence The above recombinant vector and the corresponding control vector were transiently transfected into HEK-293 cells, and the responses of the transfected cells to sweet substances were detected. The specific process is as follows: Add 5 µL of transfection reagent (Mirus TransIT-X2) to 125 µL of opti-MEM medium, mix well and set aside; then add 5 µg of plasmid (T1R2: T1R3 = 1: 1.2) to 125 µL of opti-MEM medium, mix well and set aside; mix the plasmid and transfection reagent evenly and place at room temperature for 30 minutes. Add the mixture to 3 mL of HEK-293 cells (cell density is 350,000 cells / mL), use a pipette to blow evenly, and divide the cells evenly into 96-well plates, keeping 100 µL of cells in each well. After placing the 96-well plate in an incubator and culturing for 24 hours, sweet taste sensing cells can be obtained.

[0038] Detection of the response of sweet taste receptor cells to sweet substances: Remove the culture medium, wash the cells with 100 µL PBS buffer, then add 2 µM calcium ion fluorescent probe (model: Calbryte 520 AM, manufacturer: AAT Bioquest) and incubate at room temperature in the dark for 30 min, remove the probe solution, wash the cells once with PBS, add 30 µL PBS, and place the cells in PBS solution for detection. Before the detection, remove the PBS in the well and add 30 µL of 10 mM sucrose solution. Continuously observe the changes in cell fluorescence intensity under a fluorescence microscope, and record every 5 s.

[0039] Test results such as Figure 3 As shown ( Figure 3 The comparison diagram is shown as an example of the time point with the strongest fluorescence within 1 minute from the start of detection). The sequence used removed the histidine tag and Flag tag in the N segment. The results showed that the receptor after removing the tag can better recognize sweet substances, effectively improving the sensitivity of the method. Further analysis showed that the possible reason for this phenomenon is that the sweet taste receptors T1R2 and T1R3 have similar structures. Both receptors have a Venus flytrap domain at the N-terminus for recognizing different sweet substances. The sweet taste receptor sequences used in the prior art have added histidine tags and Flag tags to the N segment of the sweet taste receptor, which may affect the receptor's recognition and binding of sweet substances.

[0040] Example 2 Optimization of recombinant vector transfection conditions This example uses the T1R2 recombinant vector and the T1R3 recombinant vector constructed in Example 1 to optimize the important conditions in the transfection process. The specific implementation operations are as follows: 1. Transformation and extraction of plasmid Thaw 50 µL competent bacteria on ice, add an appropriate amount of recombinant vector (correctly sequenced) to competent cells, mix slowly with a pipette, and place on ice for 30 min. Place competent bacteria in a 42°C water bath, heat shock for 30 seconds, and cool on ice for 5 min. Add 500 µL LB medium and culture on a shaker at 37°C for 1 hour. Take 100 µL of the cultured competent bacteria and evenly spread them on an LB culture plate with the corresponding antibiotics, and culture them upside down in a 37°C incubator for 12-18 hours.

[0041] Select a single clone colony and place it in a 50 mL culture bottle. Culture it in a shaker at 37°C for 12-18 h, and collect the bacteria by centrifugation. Extract the bacterial plasmid by alkaline lysis, quantify the obtained plasmid, dilute it to 1 µg / µL, and store it at -80°C.

[0042] 2. Condition Optimization The volume of transfection reagent (5 µL, Mirus TransIT-X2) and transfection time (24 h) were kept unchanged, and transient transfection experiments were performed using plasmid combinations with different mass ratios (the mass ratios of T1R2 recombinant vector to T1R3 recombinant vector were 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5 and 1:2, respectively), and the transfection steps were the same as described in Example 1. Subsequently, the cells were stimulated with sucrose, and the changes in intracellular calcium ion concentration were detected (the specific detection process was as described in Example 1), and it was confirmed that the optimal plasmid mass ratio was 1:1.2~2.

[0043] Keeping the amount of plasmid used (1:1.5) and transfection time (24 h) unchanged, transient transfection experiments were performed using different volumes of transfection reagent (3.5, 4.5, 5.5 and 6.5 µL), and the transfection steps were the same as described in Example 1. Subsequently, sucrose was used to stimulate the cells (the specific detection process was as described in Example 1), and the changes in intracellular calcium ion concentration were detected, confirming that the optimal amount of transfection reagent used was 4.5-5.5 µL.

[0044] The amount of plasmid used (1:1.5) and the volume of transfection reagent (5.5 µL) were kept unchanged, and the transfection time (24, 36, 48 h) was different, and the transfection steps were the same as described in Example 1. Then, sucrose was used to stimulate the cells (the specific detection process was as described in Example 1), and the changes in intracellular calcium ion concentration were detected to confirm that there was no significant change at different transfection times.

[0045] The results of cell detection after transfection with plasmid combinations of different mass ratios are as follows Figure 4 As shown, good transfection effect can be achieved when the mass ratio of the two plasmids is 1:1.2~2.

[0046] Optimize the amount of transfection reagent used and test results Figure 5 As shown in the figure, a transfection reagent dosage of 4.5~5.5 µL can achieve good transfection results.

[0047] Transfection time optimization optimization test results such as Figure 6 As shown, there was no significant change at different transfection times, but the transfection time between 24 and 36 h had little effect on the cell growth state.

[0048] Example 3 Preparation of sweet taste receptor cells 1 In this example, the recombinant vector constructed in Example 1 was used in combination with the optimized transfection conditions in Example 2 to prepare sweet taste sensing cells. The specific implementation steps are as follows: 1. Cell transfection Add 5.5 µL of transfection reagent to 125 µL of opti-MEM medium, mix well and set aside; then add 5 µg of plasmid (T1R2: T1R3 = 1: 1.2) to 125 µL of opti-MEM medium, mix well and set aside; mix the plasmid and transfection reagent evenly and place at room temperature for 30 min. Add the mixture to 3 mL of HEK-293 cells (cell density is 350,000 cells / mL), use a pipette to blow evenly, and divide the cells evenly into 96-well plates, keeping 100 µL of cells in each well. After placing the 96-well plate in an incubator and culturing for 24 h, sweet taste sensing cells 1 can be obtained.

[0049] 2. Immunoblotting to detect the expression of sweet taste receptors in cells The total protein of sweet taste receptor cells was extracted, and the expression of sweet taste receptors was detected by Western Blot to determine the expression of sweet taste receptors in cells. Figure 7 As shown in the figure, it can be seen that compared with the control group, the expression of sweet taste receptors can be observed in the transiently transfected cells.

[0050] 3. Detection of sweet taste receptor expression in cells by fluorescence quantitative PCR Total RNA from sweet taste receptor cells was extracted and cDNA was obtained by reverse transcription. Then, T1R2 and T1R3 specific primers were used to detect the expression of sweet taste receptors in cells, and ACTB was used as an internal reference gene. The sequences of fluorescent quantitative PCR primers are as follows: T1R2-F: 5'-TCCAGCCGGTGCTCTACTT-3' (as shown in SEQ ID NO. 7); T1R2-R: 5'-CCACCACACGGGAAATGTAGT-3' (as shown in SEQ ID NO. 8); T1R3-F: 5'-CCGCCTACTGCAACTACACG-3' (as shown in SEQ ID NO. 9); T1R3-R: 5'-CTAGCACCGTAGCTGACCTG-3' (as shown in SEQ ID NO. 10); ACTB-F: 5'-CATGTACGTTGCTATCCAGGC-3' (as shown in SEQ ID NO. 11); ACTB-R: 5′-CTCCTTAATGTCACGCACGAT-3′ (as shown in SEQ ID NO. 12).

[0051] Test results such as Figure 8 As shown in the figure, it can be seen that compared with the control group, the mRNA expression level of sweet taste receptor in transiently transfected cells was significantly increased.

[0052] The results of immunoblotting and fluorescence quantitative PCR detection showed that the sweet taste receptors T1R2 and T1R3 were successfully expressed in HEK-293 cells, and the sweet taste receptor cell 1 was successfully constructed.

[0053] 4. Response to sweet substances The constructed sweet taste sensing cells and cells stably expressing T1R2 protein and T1R3 protein were used to detect the response of the sweet taste cells to low concentration sucrose (2 mM sucrose) stimulation (the specific detection process is as described in Example 1). Fig. 9 As shown in the figure, it can be concluded that transiently transfected cells respond better to stimulation than stably transfected cells, which proves that transiently transfected cells have higher sensitivity.

[0054] Example 4 Preparation of sweet taste receptor cells 2 In this example, sweet taste receptor cells expressing genes encoding calcium ion indicators (nucleotide sequence as shown in SEQ ID NO.13), T1R2 and T1R3 were constructed, and the specific implementation operations were as follows: 1. Optimization of plasmid usage Keeping the transfection reagent volume (5 µL) and transfection time (24 h) unchanged, transient transfection experiments were performed using different mass ratios of genetically encoded calcium ion indicator plasmids (pGP-CMV-GCaMP6f plasmid vector containing aequorin, Miaoling Biotechnology, Cat. No.: P29749), T1R2 recombinant plasmids and T1R3 recombinant plasmids (plasmid mass ratios of 1.5:1:1.5, 2:1:1.5, 2.5:1:1.5, 3:1:1.5), and the transfection steps were the same as described in Example 1. Subsequently, the cells were stimulated with 10 mM sucrose, and the changes in intracellular calcium ion concentration were detected. The specific results are shown in Figure 1. Fig.10Combined with the optimization results of plasmid usage in Example 2, combined with Figure 3 It can be seen that the gene-encoded calcium ion indicator plasmid, T1R2 recombinant plasmid and T1R3 recombinant plasmid can all achieve good results in the ratio of (2~3):1:(1.2~2).

[0055] 2. Construction of sweet taste receptor cells encoding calcium ion indicator by co-expression gene Take 250 µL of opti-MEM medium into a sterilized 5 mL centrifuge tube, add 10 µg of gene-encoded calcium indicator plasmid, T1R2 and T1R3 mixed plasmid (3:1:1.2), and mix slowly with a pipette. Then add 11 µL of transfection reagent (Mirus TransIT-X2), mix slowly with a pipette, and after leaving at room temperature for 30 min, add the mixture to 3 mL of HEK-293 cells (cell density is 350,000 cells / mL), pipette evenly, and divide the cells evenly into 96-well plates, keeping 100 µL of cells in each well. After placing the 96-well plate in an incubator and culturing for 24 h, the co-expression gene-encoded calcium indicator sweet taste sensing cells can be obtained. The obtained sweet taste sensing cells do not need to be incubated with calcium ion fluorescent probes. Sweet substances can be used to directly stimulate cells and detect fluorescence changes.

[0056] The specific steps are as follows: remove the culture medium, wash the cells with 100 µL PBS buffer, add 30 µL PBS, and place the cells in the PBS solution for detection. Before detection, remove the PBS in the well and add 30 µL of 10 mM sucrose solution. Continuously observe the changes in cell fluorescence intensity under a fluorescence microscope and record every 5 seconds. The test results are as follows Fig.11 As shown ( Fig.11 The comparison graph shown is based on the time point with the strongest fluorescence within 1 min from the start of detection. It can be seen from the figure that the cell fluorescence intensity changes significantly before and after sucrose stimulation, indicating that sweet taste receptor cells that co-express genes encoding calcium ion indicators, T1R2 and T1R3 can be successfully used to detect sweet substances.

[0057] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing sweet taste receptor cells, characterized in that: The method comprises the following steps: transiently transfecting a recombinant vector expressing T1R2 and T1R3 into a eukaryotic cell; the mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 is 1:(1-2); the nucleotide sequence of T1R2 is shown in SEQ ID NO.1; and the nucleotide sequence of T1R3 is shown in SEQ ID NO.

2.

2. The method for preparing sweet taste receptor cells according to claim 1, characterized in that: The mass ratio of the recombinant vector expressing T1R2 to the recombinant vector expressing T1R3 is 1:(1.2-2).

3. The method for preparing sweet taste receptor cells according to claim 1 or 2, characterized in that: The transient transfection time is 24 to 36 h.

4. The method for preparing sweet taste receptor cells according to claim 3, characterized in that: The amount of transfection reagent used for the transient transfection is 4.5~5.5 μL.

5. The method for preparing sweet taste receptor cells according to claim 1, characterized in that: The preparation method comprises the steps of transiently transfecting a recombinant vector expressing a gene encoding a calcium ion indicator and a recombinant vector expressing T1R2 and T1R3 into eukaryotic cells.

6. The method for preparing sweet taste receptor cells according to claim 5, characterized in that: The genetically encoded calcium ion indicators include aequorin-based GAP series and calmodulin-based GCaMP series.

7. The method for preparing sweet taste receptor cells according to claim 5 or 6, characterized in that: The mass ratio of the recombinant vector expressing the gene encoding the calcium ion indicator, the recombinant vector expressing T1R2 and the recombinant vector expressing T1R3 is (2-3):1:(1.2-2).

8. The method for preparing sweet taste receptor cells according to any one of claims 1, 2, 5 and 6, characterized in that: The eukaryotic cells include HEK293 cells.

9. A sweet taste receptor cell prepared by the method for preparing sweet taste receptor cells according to any one of claims 1 to 8.

10. Use of the sweet taste receptor cell according to claim 9 in the rapid screening of sweet substances and the quantitative analysis of the sweetness value of sweet substances.

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