Sweet substance detection method

Abstract

The invention belongs to the field of biological detection, and particularly relates to a sweet substance detection method. The invention develops a detection method for sweet substances on the basis of a cAMP (cyclic adenosine monophosphate) way.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to a sweet substance detection method. Background Art

[0002] Sweetness is one of the five most common basic tastes in humans and is crucial to the sensory experience of food. The transmission of sweetness signals is achieved by pathways mediated by sweet taste receptors and their downstream G proteins.

[0003] Human sweet taste receptors are heterodimers composed of T1R2 and T1R3. T1R2 and T1R3 belong to the C-class family of G protein-coupled receptors (GPCRs). They have similar structures: the N-terminal extracellular domain contains a Venusflytrap module (VFT), which is then connected to seven transmembrane domains (7TMD) through a cysteine-rich domain (CRD). T1R2 and T1R3 can sense a variety of sweet molecules, including natural sugars (glucose, maltose, lactose, fructose, sucrose, etc.), artificial sugars (acesulfame potassium, sucralose, aspartame, cyclamate, etc.), and sweet proteins (brazil sweet, thaumatin, etc.). When sweet molecules bind to specific binding sites of T1R2 and T1R3, this heterodimeric receptor activates the intracellular signal transduction pathway, leading to the dissociation of downstream G protein heterotrimers. Among them, gustducin protein (a Gα subunit) causes changes in intracellular cAMP concentration by affecting the activity of adenylate cyclase (AC), while βγ dimers cause Ca2+ to increase through the PLCβ pathway. 2+ In addition, T1R2 and T1R3 can bind to Gα i subunit coupling, when the receptor is activated by sweeteners, Gα i The subunit is activated, inhibiting AC enzyme activity, resulting in a decrease in intracellular cAMP concentration.

[0004] By detecting the changes in downstream signals after the sweet taste receptors are activated, we can design biosensors that can qualitatively and quantitatively detect sweet molecules. This can provide a powerful tool for the development of new sweeteners, which is of great significance to food nutrition science, artificial intelligence perception science and medicine.

[0005] The sweet substance detection methods that have been developed mainly include electronic tongue based on electrical signals and calcium flow detection based on the cell level. Traditional sweetness detection methods such as electronic tongue require a long detection time and expensive equipment, and are also difficult to perform quantitative detection.

[0006] Split luciferase assay is one of the common methods to detect GPCR-ligand interactions. In the split luciferase assay, the luciferase protein is split into an inactive state. In the presence of cAMP, the luciferase is converted to an active state, which can catalyze the luciferase substrate to emit visible light, and then the degree of interaction between the GPCR and its ligand can be indirectly evaluated, thereby inferring the affinity or activation effect of the ligand. Summary of the invention

[0007] To solve the above problems, the present invention develops a novel detection method for sweet substances based on the cAMP pathway.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a protein combination, characterized in that the protein combination consists of a human sweet taste receptor protein, a G protein inhibitory subunit and a split luciferase protein.

[0010] In some embodiments, the human sweet taste receptor protein is T1R2 and T1R3, and the G protein inhibitory subunit is Gα i1 or Gα i2 or Gα i3 , the above-mentioned split luciferase is a luciferase with a cAMP binding domain.

[0011] In some embodiments, the amino acid sequence of the T1R2 is as shown in SEQ ID NO.1, the amino acid sequence of the T1R3 is as shown in SEQ ID NO.2, and the Gα i1 The amino acid sequence of Gα is shown in SEQ ID NO.10. i2 The amino acid sequence of Gα is shown in SEQ ID NO.11. i3 The amino acid sequence of is shown in SEQ ID NO.12, and the amino acid sequence of the above-mentioned split luciferase is shown in SEQ ID NO.13.

[0012] The present invention also provides a composition, characterized in that it comprises (1) the protein combination according to claim 1; and (2) a luciferase substrate.

[0013] In some embodiments, the luciferase substrate is luciferin or its potassium salt or sodium salt derivative.

[0014] The present invention also provides the use of the above protein combination or the above composition in detecting sweet substances.

[0015] In some embodiments, the above-mentioned sweetener is any one of aspartame, sucralose, thaumatin, brazilian sweet, saccharin, acesulfame potassium, cyclamate, neotame, steviol glycoside, sweet taste receptor inverse agonist, or a combination thereof.

[0016] The present invention also provides a method for detecting sweet substances, characterized in that it comprises the following steps:

[0017] (1) expressing the protein combination of claim 1 in a cell;

[0018] (2) adding 0.1 mM to 10 mM of the luciferase substrate described in claim 2;

[0019] (3) Incubate at 16°C to 37°C for 5 min to 4 h;

[0020] (4) adding a solution of a sweet substance to be detected;

[0021] (5) Incubate at 16°C to 37°C for 5 min to 1 h;

[0022] (6) The luminescence value is measured by an enzyme-labeled instrument, and the content of the sweet substance is calculated based on the luminescence value.

[0023] In some embodiments, the cell in the above step (1) is any one of HEK293 cells, HEK293T cells, CHO cells, and Sf9 insect cells.

[0024] In some embodiments, 1 mM of the luciferase substrate described in claim 2 is added in step (2) above.

[0025] In some embodiments, the incubation temperature in step (3) above is 23°C.

[0026] In some embodiments, the incubation time in step (3) above is 60 min to 90 min.

[0027] In some embodiments, the incubation temperature in step (5) above is 23°C.

[0028] In some embodiments, the incubation time in step (5) above is 15 min to 30 min.

[0029] The advantages and beneficial effects of the present invention are as follows: Based on the cAMP pathway, the present invention uses a suitable protein combination and composition to develop a new detection method for sweet substances. The method has good stability and is suitable for the detection of various sweet substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Map of the pCDNA3.1 backbone vector.

[0031] Figure 2 Activity test results of aspartame and sucralose based on calcium flux pathway.

[0032] Figure 3 pCDNA5-FRT / TO-Gα gustducin - Rluc8 vector map.

[0033] Figure 4 Activity detection results of aspartame and sucralose based on BRET2 pathway.

[0034] Figure 5 Activity test results of aspartame and sucralose based on cAMP pathway.

[0035] Figure 6 The effect of different incubation times after adding luciferase (A), different luciferase substrate concentrations (B), and different incubation times after adding the sweet substance to be detected (C) on the detection of sucralose via the cAMP pathway.

[0036] Figure 7 The effect of different incubation temperatures after adding luciferase (A) and different incubation temperatures after adding the sweet substance to be detected (B) on the detection of sucralose via the cAMP pathway.

[0037] Figure 8 Activity test results of aspartame and sucralose based on cAMP pathway using substrate D-Luciferin potassium salt derivative (first row) and sodium salt derivative (second row).

[0038] Fig. 9 Activity test results of aspartame and sucralose based on cAMP pathway using HEK293 stable cell line.

[0039] Fig.10 Activity test results of aspartame and sucralose using different Gα subunit proteins based on cAMP pathway.

[0040] Fig.11 Activity test results of aspartame and sucralose based on cAMP pathway using different cell lines.

[0041] Fig.12 Activity test results of various sweeteners based on cAMP pathway.

[0042] Fig.13 Activity detection results of sweet taste inverse agonists based on cAMP pathway. DETAILED DESCRIPTION

[0043] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public publications cited herein are incorporated herein by reference in their entirety.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the inventive method, step or condition, all belong to the scope of the present application. If not otherwise specified, the embodiments are according to conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 2001), or according to the conditions of the manufacturer's instructions. If not otherwise specified, the chemical reagents used in the embodiments are conventional commercial reagents, and the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0045] Example

[0046] Example 1 Sweetness activity detection based on calcium flux pathway

[0047] The sweet taste activity detection of the calcium flux pathway is a common sweet taste activity detection method based on the cellular level. The inventors first tested the technical effect of this method. The sweet taste receptors (T1R2 and T1R3, the amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively) were combined with the chimeric G protein subunit Gα 16gust44 (amino acid sequence such as SEQ ID NO.3) coupled, the subunit can cause an increase in intracellular calcium flow, and the degree of activation of the sweet taste receptor is judged by the degree of increase in calcium flow, thereby identifying the sweetness activity intensity of the sweet substance to be detected.

[0048] The specific steps are as follows:

[0049] (1) Vector construction

[0050] Synthesize T1R2 and T1R3 nucleic acid molecules (sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively) to obtain the pCDNA3.1 backbone vector (see Figure 2 for the pCDNA3.1 backbone vector). Figure 1 ), and the sweet taste receptor expression plasmids pCDNA3.1-T1R2 and pCDNA3.1-T1R3 were constructed by the Gibson Assembly method. 16gust44The nucleic acid molecule (sequence shown in SEQ ID NO.6) was used to construct the G protein subunit chimeric expression vector pCDNA3.1-Gα by Gibson Assembly 16gust44 .

[0051] (2) Cell culture and transfection

[0052] HEK293 cells were cultured in DMEM complete medium supplemented with 10% FBS and placed in a 37°C, 5% CO2 incubator. When the cell confluence reached 85% to 90%, trypsin was used for digestion, 600,000 cells were plated per well in a 6-well plate, and the culture medium was supplemented to 2 mL / well. The culture medium was shaken regularly to evenly distribute the cells and cultured in an incubator.

[0053] According to pCDNA3.1-T1R2: pCDNA3.1-T1R3: pCDNA3.1-Gα 16gust44 =1:1:1 (total 2.5 μg / well) co-transfected into cells. 24 hours after transfection, cells were spread into PLL-coated black bottom transparent 96-well plates and cultured in an incubator.

[0054] (3) Calcium flow detection

[0055] After 16-24 hours, discard the culture medium, wash once with DPBS, and add calcium ion staining working solution at 50μL / well. Working solution configuration: 2.5μg / mL Fluo-4AM mixed with 0.25% Pluronic F-127 dissolved in DPBS. Incubate in the dark for 60 minutes, wash three times with DPBS, and incubate the 96-well plate in the dark at room temperature for 30 minutes.

[0056] The calcium flux changes were recorded using a Flexstation3 multifunctional microplate reader, and RFU was recorded every 2 seconds for each well for a total of 200 seconds. The ligand aspartame or sucralose solution was added at 30 seconds, and the peak RFU after the addition of the ligand was recorded as F, and the subsequent baseline RFU was recorded as F0. ΔF = F-F0, and ΔF / F was used as the degree of receptor response to the ligand. The data were analyzed using "Inhibitor vs Response" or "log (inhibitor) vs Response" in GraphPad Prism. The results of multiple experiments showed that the standard deviation of the fluorescence change value corresponding to each concentration of aspartame and sucralose was large, and the R of the entire fitting curve was 2 Less than 0.9( Figure 2), and the results of multiple batches of repetitions also showed the same problem (Table 1), indicating that the test results of this method have large errors, cannot be used for more accurate detection, and cannot be used more widely.

[0057] Table 1 Results of different batches of sucralose activity assay based on calcium flux pathway

[0058]

[0059] Example 2 Sweetness activity detection based on bioluminescence resonance energy transfer (BRET2)

[0060] GPCR signaling pathways can also be detected by BRET2, so the inventors also tested the technical effect of this method. gustducin (amino acid sequence as shown in SEQ ID NO.7) can be combined with Gβ3 (amino acid sequence as shown in SEQ ID NO.8) and Gγ 13 (amino acid sequence as shown in SEQ ID NO.9) to form a heterotrimer. When the sweet taste receptor is activated, Gα gustducin β3γ 13 Dimer dissociation. The degree of dissociation can be detected by BRET2, so that the degree of sweet taste receptor activation can be known, and finally the activity of sweet substances can be detected. The specific operation steps are as follows:

[0061] (1) Vector construction

[0062] Synthetic Gα gustducin , Gβ3 and Gγ 13 Nucleic acid molecules (sequences are shown in SEQ ID NO.7), construct G protein subunit chimeric expression plasmid pCDNA5-FRT / TO-Gα gustducin -Rluc8 (vector map see Figure 3 ), pCDNA3.1-Gβ3 and pCDNA3.1-Gγ 13 -GFP2.

[0063] (2) Cell culture and transfection

[0064] According to the steps in Example 1, cells were cultured and the pCDNA3.1-T1R2: pCDNA3.1-T1R3: pCDNA5-FRT / TO-G αgustducin- Rluc8:

[0065] pCDNA3.1-Gβ3:pCDNA3.1-Gγ 13-GFP2=1:1:1:1:1 (total 2.5 μg / well) was co-transfected into cells. 24 hours after transfection, the cells were spread into a PLL-coated white opaque 96-well plate and placed in an incubator for culture.

[0066] (3) BRET2 detection

[0067] After 16-24h, discard the culture medium and add the working solution at 50μL / well. Working solution preparation: DPBS dilutes coelenterazine 400a to a final concentration of 5μM. After standing for 5min, add 25μL of aspartame or sucralose of different concentrations and incubate for 5min. Use Mithras LB940 multifunctional microplate reader to detect the emission light of 395nm and 510nm wavelengths. The reading speed is 1s per well, and the detection is repeated 3 times. The result of the third plate reading is used as the final analysis result. Calculate with BRET2=Em510nm / Em395nm, and use "Inhibitor vs Response" or "log(inhibitor)vs Response" in GraphPad Prism to analyze the data. The results showed that there was no obvious dose-effect relationship between sweetener concentration and BRET2 ( Figure 4 ), indicating that the dissociation degree of G protein trimer did not increase due to the enhancement of sweetener activity, and the activity of sweeteners could not be detected by this method.

[0068] Example 3 Sweetness Activity Detection Based on cAMP Pathway

[0069] The inventors further attempted a sweet substance detection method based on the cAMP pathway. Since sweet taste receptors (such as T1R2 and T1R3) can interact with the inhibitory G protein α subunit Gα i (including Gα i1 , Gα i2 and Gα i3 etc., the amino acid sequences are shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively) and Gα i It can inhibit the aggregation of intracellular cAMP. After the sweet taste receptor is activated, its coupled Gαi subunit dissociates and acts as an inhibitor of AC enzyme (adenylate cyclase), inhibiting the aggregation of intracellular cAMP. The degree of activation of the sweet taste receptor can be analyzed by the level of reduction in the intracellular cAMP concentration, thereby detecting the activity of the sweet substance to be tested. The specific operation steps are as follows:

[0070] (1) Vector construction

[0071] Synthetic Gα i1and cAMP-Luc nucleic acid molecules (sequences are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively), to construct pCDNA3.1-Gα i1 and pCDNA3.1-cAMP-Luc vector.

[0072] (2) Cell culture and transfection

[0073] According to the steps in Example 1, cells were cultured and co-transfected with pCDNA3.1-T1R2, pCDNA3.1-T1R3, and pCDNA3.1-Gα. i1 and pCDNA3.1-cAMP-Luc, add them to 250μL Opti-Mem at a ratio of 1:1:1:7, then add 10μL PEI, mix gently with a pipette, let stand for 10-15min, gently disperse the transfection complex drop by drop into the well plate, shake gently and place in the incubator. 24h after transfection, disperse the cells into a 96-well plate and place in the incubator for culture.

[0074] (3) Biochemiluminescence detection based on cAMP pathway

[0075] After 18-24 hours of plating in the 96-well plate, the culture medium was discarded and 50 μL / well of 2 mM luciferase substrate luciferin (D-Luciferin) (MedChemExpress brand, catalog number HY-12591A) working solution was added. After incubation at 25°C for 2 hours, 3× ligand solution was added to the well plate at 25 μL / well. After incubation at 25°C for another hour, a Tecan Spark microplate reader was used to scan the full wavelength luminescence to read the luminescence value. The specific settings were: 25°C, 1s / well. Use "log (inhibitor) vs Response" in GraphPad Prism to analyze the data. The results of multiple experiments using this method showed that the standard deviation of the fluorescence change value corresponding to each concentration of aspartame and sucralose was very small, and the R of the entire fitting curve was 2 Very high value ( Figure 5 ), the results of multiple batches of repetitions also showed strong stability (Table 2), indicating that the technical effect of this method is better than that of the methods in Example 1 and Example 2.

[0076] Table 2 Results of different batches of sucralose activity assay based on cAMP pathway

[0077]

[0078] The inventors further optimized the sweetness activity detection method of the cAMP pathway. Since luciferase has a great influence on the detection signal, it determines the final detection result. Therefore, the test optimization was mainly carried out from the incubation time after adding luciferase, the incubation temperature range after adding luciferase, the luciferase substrate concentration range, the incubation temperature range after adding the ligand to be detected, and the incubation time range after adding the ligand to be detected. The results show that:

[0079] 1. The incubation time after adding luciferase is between 5 minutes and 4 hours. When the incubation time is less than 5 minutes, the luminescence intensity is in a high-speed increasing state, and subsequent detection at this time may have a greater impact on the results; when the incubation time exceeds 4 hours, the luminescence intensity decreases rapidly with time, and it is not suitable for subsequent detection. The optimal incubation time range is 60 to 90 minutes, at which time the luminescence intensity is stable, with small fluctuations, and the impact on subsequent detection results is minimal.

[0080] 2. After adding luciferase, the incubation temperature range is between 16℃ and 37℃. When the incubation temperature is lower than 16℃, the luciferase activity is inhibited, the luminescence intensity is low, and it cannot meet the requirements of subsequent detection experiments; when the incubation temperature exceeds 37℃, the luciferase is rapidly inactivated at a higher ambient temperature and cannot continuously provide the luminescence intensity required for detection. The optimal incubation temperature is 23℃, at which the luminescence intensity is higher and the fluctuation is minimal, which has the least impact on subsequent detection results.

[0081] 3. The concentration range of luciferase substrate is between 0.1mM and 10mM. When the concentration of luciferase substrate is lower than 0.1mM, the luminescence intensity is not enough for subsequent detection; when the concentration of luciferase substrate is higher than 10mM, the luminescence intensity enters a plateau and does not increase. The optimal concentration is 1mM, at which the luminescence intensity is high enough to meet the requirements of subsequent detection, and the moderate concentration of substrate has little interference with subsequent detection.

[0082] 4. After adding the ligand to be detected, the incubation temperature range is between 16℃ and 37℃. When the incubation temperature is lower than 16℃, the luciferase activity is inhibited, the luminescence intensity is low, and the time required for the ligand to reach the maximum inhibitory effect is longer, which has a greater impact on the accuracy of the results; when the incubation temperature exceeds 37℃, the luciferase is rapidly inactivated at a higher ambient temperature, and the luminescence intensity continues to decrease, and it is unable to continuously provide the luminescence intensity required for detection. The optimal incubation temperature is 23℃, at which the luminescence intensity is higher, and the ligand can quickly reach the maximum inhibitory effect on the concentration of cAMP, and the luminescence intensity tends to be stable after reaching the maximum inhibitory effect, which has little effect on the results.

[0083] 5. After adding the ligand to be detected, the incubation time ranges from 5min to 60min. When the incubation time is less than 5min, the concentration of the ligand on cAMP has not reached the maximum inhibitory effect. At this time, subsequent detection will have a greater impact on the accuracy of the results; when the incubation time exceeds 1h, the receptor may be desensitized when a high concentration of ligand exists for a long time, and the accuracy of the test results will be affected. The optimal incubation time range is 15-30min, at which time the concentration of the ligand on cAMP has reached the maximum inhibitory effect, and the subsequent luminescence intensity is stable and fluctuates slightly, which has the least impact on the subsequent test results.

[0084] According to the above test results, the operation of step (3) in the above detection steps can be:

[0085] After 18-24 hours of plating on the 96-well plate, discard the culture medium and add 50 μL / well of 0.1-10 mM luciferase substrate luciferin (D-Luciferin) (MedChemExpress, catalog number HY-12591A) working solution. After incubation at 16-37°C for 5 min-4 h, add 3× ligand solution to the well plate at 25 μL / well. After incubation at 16-37°C for another 5 min-1 h, use a TecanSpark microplate reader to scan the full wavelength luminescence to read the luminescence value. The specific settings are: 25°C, 1s / well. Use "log (inhibitor) vs Response" in GraphPad Prism to analyze the data.

[0086] Example 4 Detection of sweet substances using D-Luciferin derivatives and stable cell lines

[0087] The inventors further tested the technical effect of detecting sweet substances using D-Luciferin derivatives and stably transfected cell lines based on the method of Example 3 above.

[0088] 1. The luciferase substrate D-Luciferin was replaced with its potassium salt derivative (MedChemExpress, Catalog No. HY-12591B) and sodium salt derivative (MedChemExpress, Catalog No. HY-12591) to identify the activity of aspartame and sucralose. The results showed that D-Luciferin and its potassium and sodium salt derivatives can be catalyzed as split luciferase substrates and produce different intensities of biochemiluminescence according to the different cAMP concentrations ( Figure 8 ).

[0089] 2. Use stable cell lines instead of plasmid transient transfection to detect sweet substances. The specific steps are as follows:

[0090] (1) Construction of HEK293 cell line stably co-expressing T1R2 / T1R3 sweet taste receptor and split luciferase cAMP-Luc

[0091] A. Construction of Lentiviral Expression Vector

[0092] The T1R2 gene fragment was cloned into the lentiviral vector pLVX-T1R2-HygR with hygromycin B resistance using Gibson Assembly, and the hygromycin B resistance gene on the lentiviral vector pLVX-HygR was replaced with the puromycin resistance gene Puro and the bleomycin resistance gene Zeo using Gibson Assembly to obtain the recombinant vectors pLVX-Puro and pLVX-Zeo. The T1R3 gene fragment and cAMP-Luc fragment were cloned into the vectors pLVX-Puro and pLVX-Zeo, respectively, to obtain the recombinant vectors pLVX-T1R3-Puro and pLVX-cAMP-Luc-Zeo.

[0093] B. Preparation of Lentivirus

[0094] According to the cell culture and co-transfection steps in Example 1, cells were co-transfected in three 6-well plates.

[0095] i) pLVX-T1R2-Puro, PxpaX2, PMD2g; ii) pLVX-T1R3-Puro, PxpaX2, PMD2g; iii) pLVX-cAMP-Luc-Zeo, PxpaX2, PMD2g. The transfection ratio is pLVX: PxpaX2: PMD2g = 4:3:1.

[0096] 8-12h after transfection, replace the supernatant containing transfection reagent with DMEM complete medium. 48h after transfection, collect the virus-containing culture supernatant in a 5mL tube, centrifuge at 1000g for 5min to remove cell debris and impurities, and then filter using a syringe and 0.45μm filter. The collected virus supernatant is used for subsequent infection or stored at -80℃ for long-term storage.

[0097] C. Lentivirus infection of cells

[0098] According to the cell culture steps in Implementation Example 1, when the cells in the 6-well plate grow to the logarithmic phase, that is, when the confluence is 30% to 50%, the virus mixture is added to the target cells at a ratio of virus solution: fresh culture medium = 1:1, and the cells are shaken and placed in an incubator for culture.

[0099] D. Screening of cell lines stably expressing T1R2

[0100] Before screening the stable cell line, the killing curve of different antibiotics on the cells was determined, and finally the concentrations of the antibiotics used for screening were determined to be 400μg / mL Hygromycin B, 5μg / mL Puromycin and 300μg / mL Zeocin. The target cells were infected with a mixture of lentiviruses carrying the T1R2 gene. The cells infected with the virus for 72 hours were digested and plated on a 6-well plate. The next day, after the cells adhered to the wall, DMEM complete medium containing 400μg / mL Hygromycin B was replaced. The complete medium with antibodies was replaced every two days, and the cell density reached 90% before passage. After the cells stabilized, DMEM complete medium containing 200μg / mL Hygromycin B was used to maintain the growth of the T1R2 cell line.

[0101] E. Screening of cell lines stably expressing T1R2 / T1R3

[0102] According to step (4), the stably expressing T1R2 cell line was infected with a lentivirus mixture carrying the T1R3 gene, and screened using a complete medium containing 5 μg / ml Puromycin. After the cells were stabilized, the growth of the stably expressing T1R2 / T1R3 cell line was maintained using a complete medium containing 200 μg / mL Hygromycin B and 2.5 μg / mL Puromycin.

[0103] F. Screening of cell lines stably expressing T1R2 / T1R3 / cAMP-Luc

[0104] According to step (4) or (5), the cell line stably expressing T1R2 / T1R3 is infected with a lentivirus mixture carrying the cAMP-Luc gene, and screened using a complete medium containing 300 μg / mL Zeocin. After the cells are stable, a complete medium containing 200 μg / mL Hygromycin B, 2.5 μg / mL Puromycin and 150 μg / mL Zeocin is used to maintain the growth of the cell line stably expressing T1R2 / T1R3 / cAMP-Luc.

[0105] (2) Detection of sweeteners in cell lines stably expressing T1R2 / T1R3 / cAMP-Luc

[0106] The cell line stably expressing T1R2 / T1R3 / cAMP-Luc was cultured in complete medium supplemented with 10% FBS, 200μg / mL HygromycinB, 2.5μg / mL Puromycin, and 150μg / mL Zeocin, and digested with trypsin when the confluence reached 85% to 95%. The cells were plated at a density of 50,000 / well in a 96-well plate and cultured in an incubator.

[0107] After 24 hours of plating, the typical sweeteners aspartame and sucralose were detected according to the steps of biochemiluminescence detection in Example 3. The results showed that the stable cell line can also detect sweet substances ( Fig. 9 ), and compared with plasmid transient transfection, after the sweet taste receptors in the stable cell line are activated by sweet substances, the change in downstream cAMP is greater and the detection sensitivity is higher.

[0108] Example 5 Detection of sweet substances using different Gα subunits

[0109] The inventors further tested the technical effect of using different Gα subunits on the detection of sweet substances. The Gα subunits tested in this embodiment include Gα s (Another subunit that mediates cAMP, with an opposite effect to Gαi, which increases the intracellular cAMP concentration, and its amino acid sequence is shown in SEQ ID NO.17), Gα i1 , Gα i2 and Gα i3 .

[0110] According to the method of Example 3, pCDNA3.1-Gα i1 Gα in plasmid i1 Replaced by Gα i2 (sequence as shown in SEQ ID NO.15) or Gα i3 (sequence as shown in SEQ ID NO.16) or not transfected with Gα i Subunits rely solely on endogenous Gα in HEK293 cells i The results showed that whether using (transiently transfected) Gα i1 ,Gα i2 , Gα i3 Endogenous Gα i subunits, both of which can detect sweet substances, and Gα s Subunits cannot detect sweet substances ( Fig.10 ).

[0111] Example 6 Detection of sweet substances using different cell lines

[0112] The inventors further tested the technical effect of using different cell lines for sweet substance detection. The cell lines tested in this example include HEK293T cells, CHO cells, and Sf9 insect cells, and the sweeteners detected are aspartame and sucralose.

[0113] The specific steps for using Sf9 insect cells for detection are as follows:

[0114] Sf9 insect cells were plated in 6-well plates at a seeding density of 5 × 10 4 viable cells / cm 2 , and grown as a monolayer in Sf900 IISFM medium in a non-humidified, ambient air-conditioned incubator at 28°C.

[0115] The Bac-to-Bac baculovirus expression system was used to express T1R2, T1R3, and Gα i3 The baculovirus solution containing the cAMP-Luc gene was then used to infect Sf9 cells.

[0116] 48 h after infection, the typical sweeteners aspartame and sucralose were tested according to the steps of Implementation Case 3.

[0117] HEK293T cells and CHO cells refer to the steps of Example 3.

[0118] The results showed that the above cell lines can detect sweet substances ( Fig.11 ).

[0119] Applicability of the technical solution of Example 7

[0120] In order to clarify the applicability of the technical solution, the inventors tested the above method on typical small molecule sweeteners aspartame, sucralose and macromolecular sweeteners thaumatin and brazilian sweeteners, as well as other sweeteners such as saccharin, acesulfame K, cyclamate, stevioside, and sweetness inhibitor lactisole (sweetness receptor inverse agonist). The results show that this method can effectively detect various sweet substances and sweetness inhibitors ( Fig.12 , Fig.13 ).

[0121] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A protein combination, characterized in that The protein combination consists of human sweet taste receptor protein, G protein inhibitory subunit and split luciferase protein; Optionally, the human sweet taste receptor proteins are T1R2 and T1R3, and the G protein inhibitory subunit is Gα i1 or Gα i2 or Gα i3 , the split luciferase is a luciferase with a cAMP binding domain; Optionally, the amino acid sequence of T1R2 is as shown in SEQ ID NO.1, the amino acid sequence of T1R3 is as shown in SEQ ID NO.2, and the Gα i1 The amino acid sequence of Gα is shown in SEQ ID NO.

10. i2 The amino acid sequence of Gα is shown in SEQ ID NO.

11. i3 The amino acid sequence of is shown in SEQ ID NO.12, and the amino acid sequence of the split luciferase is shown in SEQ ID NO.

13.

2. A composition, characterized in that The method comprises: (1) the protein combination according to claim 1; (2) a luciferase substrate; Optionally, the luciferase substrate is luciferin or its potassium salt or sodium salt derivative.

3. Use of the protein combination according to claim 1 or the composition according to claim 2 in detecting sweet substances; Optionally, the sweetener is any one of aspartame, sucralose, thaumatin, brazilian sweetener, saccharin, acesulfame potassium, cyclamate, neotame, steviol glycoside, sweet taste receptor inverse agonist, or a combination thereof.

4. A method for detecting sweet substances, characterized in that: The steps include: (1) expressing the protein combination of claim 1 in a cell; (2) adding 0.1 mM to 10 mM of the luciferase substrate described in claim 2; (3) Incubate at 16°C to 37°C for 5 min to 4 h; (4) Add the sweet substance solution to be tested (5) Incubate at 16°C to 37°C for 5 min to 1 h; (6) The luminescence value is measured by an enzyme-labeled instrument, and the content of the sweet substance is calculated based on the luminescence value.

5. The method according to claim 4, characterized in that The cell in step (1) is any one of HEK293 cells, HEK293T cells, CHO cells, and Sf9 insect cells.

6. The method according to claim 4, characterized in that In the step (2), 1 mM of the luciferase substrate described in claim 2 is added.

7. The method according to claim 4, characterized in that The incubation temperature in step (3) is 23°C.

8. The method according to claim 4, characterized in that The incubation time in step (3) is 60 min to 90 min.

9. The method according to claim 4, characterized in that The incubation temperature in step (5) is 23°C.

10. The method according to claim 4, characterized in that The incubation time in step (5) is 15 min to 30 min.

Images