Specific fluorescent probe and application thereof in detecting binding capacity of compound and retinoic acid X receptor alpha

Abstract

The invention discloses a specific fluorescent probe and application thereof in detecting the binding capacity of a compound and a retinoic acid X receptor alpha, and belongs to the technical field of biological analysis. The specific fluorescent probe is a compound as shown in a formula (I), wherein R is an inorganic or organic group capable of generating a fluorescence detection signal; on the basis of a crystal structure of a retinoic acid X receptor alpha and small molecule 2, 4-di-tert-butylphenol, a compound shown as a formula (I) is designed as a specific fluorescent probe, and a receptor binding pocket is precisely targeted, so that high-specificity binding is realized; in combination with fluorescence polarization detection analysis, the method can quantitatively evaluate the direct interaction between a sample to be detected and the retinoic acid X receptor alpha, ensures clear binding sites and strong specificity, and has the advantages of low experimental cost, simplicity and convenience in operation, no toxicity and the like. The invention provides an efficient screening tool for drug research and development of the targeted retinoic acid X receptor alpha.

Description

TECHNICAL FIELD

[0001] The present application relates to a specific fluorescent probe and its application in detecting the binding ability of a compound to retinoid X receptor alpha, belonging to the technical field of biological analysis. BACKGROUND

[0002] Retinoid X receptor alpha (RXRα) as an important member of the nuclear receptor superfamily plays a key role in physiological and pathological processes such as lipid metabolism, cell differentiation, immune regulation and cancer occurrence; its function mainly depends on forming homodimer or heterodimer with other nuclear receptors to regulate the transcriptional expression of downstream genes. In the form of homodimer, RXRα can directly bind to its specific ligand such as 9-cis retinoic acid and activate a series of target genes; while in the form of heterodimer, RXRα can interact with multiple nuclear receptor family members, significantly expanding its physiological function; relevant studies have shown that RXRα forms a dimer with peroxisome proliferator-activated receptors (PPAR) to regulate fatty acid metabolism and insulin sensitivity, and plays an important role in diabetes and lipid metabolism disorders; forms a dimer with thyroid hormone receptor (TR) to mediate thyroid hormone signaling and maintain the homeostasis of body growth and energy metabolism; and after binding with liver X receptor (LXR), it plays a key role in the regulation of cholesterol metabolism and atherosclerosis; in recent years, some relevant studies have pointed out that although some environmental endocrine disruptors do not directly act on related receptors (such as peroxisome proliferator-activated receptors and thyroid hormone receptors), they can indirectly affect RXRα-dependent signaling pathways by acting on RXRα; therefore, the screening of RXRα ligands is not only a key step in the development of targeted drugs for metabolic diseases and neurodegenerative diseases, but also an important means to evaluate the toxicity of environmental endocrine disruptors.

[0003] The radioactive isotope labeling method (such as 3H-9-cis retinoic acid binding experiment) has been regarded as the "gold standard" for detecting RXRα ligands for a long time; however, the inherent defects of this method, including strict waste disposal requirements and potential health risks to operators, make it difficult to adapt to high-throughput drug screening platforms; in addition, the synthesis and purification of radioactive labeled ligands are costly, which limits their application in resource-limited laboratories; in recent years, time-resolved fluorescence resonance energy transfer (TR-FRET) technology has realized detection through energy transfer between terbium (Tb) labeled antibodies and fluorescein labeled auxiliary activation peptides, for example, the LanthaScreen™ RXR detection kit of Thermo Fisher Company, although it avoids radioactive risks, but it relies on expensive rare earth element labeled reagents, which is costly in terms of reagent cost, expensive in terms of detection equipment, and cannot realize high-throughput detection, limiting its large-scale popularization and application.

[0004] In addition, the fluorescence quenching method (FQM) is based on the spontaneous fluorescence characteristics of the tryptophan residues in the RXR ligand binding domain (LBD), and evaluates the binding ability through the fluorescence intensity change caused by ligand binding; however, the excitation wavelength and emission wavelength of tryptophan are beyond the detection range of the conventional enzyme marker, forcing the experiment to rely on special ultraviolet detection equipment, which significantly increases the technical threshold; more importantly, this method has low signal-to-noise ratio, limited detection ability for weakly bound ligands, and is easily interfered by other fluorescent substances (such as serum proteins) in the sample; in 2021, the team of Shoya Yamada developed the chrysin derivative probe CU-6PMN, which realized non-radioactive detection through fluorescence enhancement effect; but when using this probe to screen natural products, it will be interfered by other components in the natural products, which may affect the accuracy of the results.

[0005] However, the existing RXRα ligand screening technology is limited by multiple challenges such as sensitivity, safety or cost, and it is difficult to meet the demand for efficient and accurate screening methods in modern scientific research, so it is urgent to develop a specific fluorescent probe for detecting the binding ability of compounds to retinoic acid X receptor alpha (RXRα) and its application, which has high sensitivity, low cost, high throughput, convenient operation (compatible with conventional enzyme markers) and anti-interference ability, to break through the current technical bottleneck. SUMMARY

[0006] To solve or partially solve the problem of detecting the binding ability of the test compound to RXRα at the molecular level under the conditions of low cost, simple operation process, wide application range and high throughput.

[0007] The object of the present application is to provide a specific fluorescent probe, which is a compound represented by formula (I); Formula (I) R is an inorganic or organic group that generates a fluorescent detection signal.

[0008] As a further preferred embodiment of the present application, the specific fluorescent probe is a compound represented by formula (II); Formula (II) Another object of the present application is to provide the application of the specific fluorescent probe in detecting the binding ability of compounds to retinoic acid X receptor alpha (RXRα); the specific fluorescent probe can be used to detect the binding ability of compounds to RXRα at the molecular level.

[0009] The ability of the sample to be tested to compete with the specific fluorescent probe for binding to RXRα is detected, so as to obtain the binding ability of the sample to be tested to RXRα, and the specific process is as follows: the sample to be tested, the specific fluorescent probe and RXRα protein are co-incubated, and a multifunctional enzyme label instrument is used to detect the fluorescence polarization value, and the IC 50 The concentration value is used to judge the binding ability of the test compound to retinoic acid X receptor alpha (RXRα).

[0010] Preferably, the co-incubation condition of the present application can be specifically as follows: incubation at room temperature for 5-30 min.

[0011] Preferably, the initial time of the co-incubation of the present application is as follows: the concentration of the specific fluorescent probe can be 20-200 nmol / L (such as 20 nmol / L); the initial concentration of the sample to be tested can be 0-20 μmol / L (such as 0.31 μmol / L, 0.62 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L, 10 μmol / L, 20 μmol / L); and the concentration of the RXRα protein can be 100-6400 nmol / L (such as 100 nmol / L).

[0012] Preferably, the specific method of the multifunctional enzyme label instrument detection of the present application is as follows: the fluorescence polarization of the FITC channel of the sample after co-incubation is detected, and the obtained fluorescence polarization average value and the concentration of the sample to be tested are plotted and analyzed.

[0013] Preferably, in the method of the present application, the Graphpad Prism software can be used to plot the RXRα protein binding experiment results, so as to obtain the binding curve of the TTBP-3C Link-FITC probe and the RXRα protein, and the binding constant 248.9 nmol / L is obtained, and the binding constant can be used to judge that the TTBP-3C Link-FITC probe has high binding ability to the RXRα protein.

[0014] Preferably, in the method of the present application, the fluorescence polarization value can be plotted against the concentration of the sample to be tested, so as to obtain a competition curve; and the Origin software can be used to perform Sigmoidal fitting on the competition curve, so as to obtain the IC 50 concentration value of the sample to be tested. 50 The smaller the IC

[0015] Preferably, the RXRα protein of the present application is synthesized by OriGene Technologies Company.

[0016] Preferably, the RXRα of the present application is a protein as shown in (a1) or (a2) or (a3) below: (a1) a protein with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing; (a2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the protein shown in SEQ ID NO: 1 in the sequence listing; (a3) a protein obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and having the same function.

[0017] The specific names of the compounds are as follows: compound 1 is 2,4,6-tri-tert-butylphenol, compound 2 is tert-butyl 2-bromoethylcarbamate, compound 3 is tert-butyl (2-(2,4,6-tri-tert-butylphenoxy)ethyl)carbamate, compound 4 is 2-(2,4,6-tri-tert-butylphenoxy)ethan-1-amine, and compound 5 is 3',6'-dihydroxy-5-thiocyanato-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one, the structural formula of which is shown as Figure 1 Compound 1, compound 2 and compound 5 are commercially available products, and compound 3 and compound 4 are synthetic intermediates.

[0018] The preparation process of the specific fluorescent probe TTBP-3C Link-FITC of the present application is as follows: compound 1 is dissolved in DMSO, sodium hydride is added, stirred at room temperature for 30 min, then compound 2 is added, heated to 100℃ and reacted for 5 h; after cooling, 10 mL of water is added for dilution, extracted with ethyl acetate for three times, the organic phases are combined, dried with anhydrous calcium chloride, and the organic solvent is removed by reduced pressure distillation; then separated by column chromatography to obtain compound 3; compound 3 is dissolved in dichloromethane, 1 mol / L hydrochloric acid in dioxane is added, stirred for 3 h; the organic solvent is removed by reduced pressure distillation, and the remaining material is the hydrochloride of compound 4; compound 5 is dissolved in DMSO, triethylamine and the hydrochloride of compound 4 are added, stirred at 50℃ for 3 h; after cooling, 10 mL of water is added for dilution, extracted with ethyl acetate for three times, the organic phases are combined, dried with anhydrous calcium chloride, and the organic solvent is removed by reduced pressure distillation; then separated by column chromatography to obtain the probe TTBP-3C Link-FITC, the structure of which is shown in formula (I), and each raw material is added according to the stoichiometric ratio.

[0019] Formula (I) The specific fluorescent probe is used to compare the binding ability of compound LG100268 and TBBPA to RXRα: The specific fluorescence probe is combined with RXR alpha protein of different concentrations, and appropriate RXR alpha protein concentration is selected, and experiments show that more probes are combined with RXR alpha protein as the RXR alpha protein concentration increases, and therefore the fluorescence polarization value presents a gradually rising trend; the fluorescence polarization value is plotted with the concentration of the specific fluorescence probe TTBP-3C Link-FITC to obtain a binding curve; the Origin software is used for Sigmoidal fitting of the binding curve, and the GraphPad software is used to calculate the binding constant of the specific fluorescence probe TTBP-3C Link-FITC and RXR alpha protein; the specific fluorescence probe is used for fluorescence polarization detection of the binding ability of different concentrations of LG100268 and TBBPA and RXR alpha, the average fluorescence polarization value is plotted with the concentration of LG100268 and TBBPA for statistical analysis; the Origin software is used for Sigmoidal fitting of the competition curve, and the IC 50 value of LG100268 and TBBPA is obtained. 50 By comparing the IC 50 values of LG100268 and TBBPA, it can be judged that the binding ability of TBBPA and RXR alpha is 1 / 15 of that of LG100268.

[0020] Mechanism of the present application: When only the TTBP-3C Link-FITC fluorescence probe exists in the detection system, the probe is in a free state, and the fluorescence polarization value is low. When RXR alpha protein is added to the system, the fluorescence polarization value of the probe combined with RXR alpha protein is high. When the test compound is further added to the system, if the compound can be combined with RXR alpha protein, the TTBP-3C Link-FITC fluorescence probe will be competed down from the protein, so that it returns to the free state, and the fluorescence polarization value will decrease. Therefore, the binding amount of TTBP-3C Link-FITC fluorescence probe and RXR alpha protein can be judged by the change of the fluorescence polarization value.

[0021] Advantages of the present application: (1) The method of the present application only needs to use RXR alpha and a specific fluorescence probe, and the experimental cost is low.

[0022] (2) The method of the present application is based on the fluorescence polarization detection mode for signal detection, and ordinary multifunctional enzyme labelers can be equipped with this function, so that the method is easy to use.

[0023] (3) The method of the present application can use a multifunctional enzyme labeler to detect in a 384-well plate, and high-throughput detection of various compounds can be realized.

[0024] (4) The method of the present application can detect the binding ability of the compound and RXR alpha at the molecular level, and has the advantages of good specificity, simple operation, etc.

[0025] The method of the present application can evaluate the binding ability of a compound to RXRa, which has important application value in evaluating the human health risk of a compound. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Chemical reaction flow chart for preparing specific fluorescent probe TTBP-3C Link-FITC; Figure 2 NMR detection result of specific fluorescent probe TTBP-3C Link-FITC; Figure 3 HPLC detection result of specific fluorescent probe TTBP-3C Link-FITC; Figure 4 MS detection result of specific fluorescent probe TTBP-3C Link-FITC; Figure 5 Binding curve of specific fluorescent probe TTBP-3C Link-FITC and RXRa; Figure 6 Detection of binding ability of LG100268 to RXRa using specific fluorescent probe TTBP-3C Link-FITC; Figure 7 Detection of binding ability of TBBPA to RXRa using specific fluorescent probe TTBP-3C Link-FITC. DETAILED DESCRIPTION

[0027] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the scope of protection of the present application is not limited to the described content.

[0028] In the following examples, the experimental methods are conventional methods, unless otherwise specified.

[0029] In the following examples, the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0030] In the following examples, three parallel repeated experiments were set, and the results were averaged.

[0031] In the following examples, the composition of 50 mmol / L potassium phosphate buffer is: 50 mmol / L potassium phosphate, 150 mmol / L potassium chloride, 0.5 mmol / L ethylenediaminetetraacetic acid (EDTA), pH 7.5.

[0032] The specific fluorescent probe was synthesized by Beijing Cabintec Technology Co., Ltd. (TTBP-3C Link-FITC specific fluorescent probe). The multifunctional enzyme marker was SpectraMax i3x of Molecular Devices Company of the United States; and the 384-hole plate was a product of Corning Company (model number 4511).

[0033] Example 1 The specific fluorescent probe TTBP-3C Link-FITC was prepared by the following specific steps: (1) Synthesis of compound 3 Compound 1 (1 g, 3.8 mmol) was dissolved in DMSO (10 mL), sodium hydride (0.3 g, 7.6 mmol) was added, and stirring was carried out at room temperature for 30 minutes; then compound 2 (4.2 g, 19 mmol) was added, and heating was carried out to 100 DEG C for 5 h; after cooling, 10 mL of water was added for dilution, extraction was carried out with ethyl acetate for three times, the organic phases were combined, dried with anhydrous calcium chloride, and the organic solvent was removed by reduced pressure distillation; then separation was carried out by column chromatography (eluent: mixture of n-hexane (Hexane) and ethyl acetate (EtOAc) in a volume ratio of 20:1) to obtain compound 3 (650 mg, purity about 42%).

[0034] (2) Synthesis of compound 4 Compound 3 (650 mg, 1.6 mmol) was dissolved in dichloromethane (10 mL), 1 mol / L hydrochloric acid dioxane (10 mL) was added, and stirring was carried out for 3 h; the organic solvent was removed by reduced pressure distillation, and the residue (480 mg) was the hydrochloride of compound 4, which could be directly used in the next reaction.

[0035] (3) Synthesis of probe TTBP-3C Link-FITC Compound 5 (200 mg, 0.51 mmol) was dissolved in DMSO (5 mL), triethylamine (155 mg, 1.53 mmol) and the hydrochloride of compound 4 (207 mg, 0.61 mmol) were added, and stirring was carried out at 50 DEG C for 3 h; after cooling, 10 mL of water was added for dilution, extraction was carried out with ethyl acetate for three times, the organic phases were combined, dried with anhydrous calcium chloride, and the organic solvent was removed by reduced pressure distillation; then separation was carried out by column chromatography (eluent: mixture of CH2Cl2 and base (MeOH) in a volume ratio of 30:1) to obtain probe TTBP-3C Link-FITC 180 mg, purity about 51%, (Chinese full name: 1-(3', 6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1, 9'-xanthene]-5-yl)-3-(2-(2, 4, 6-tri-tert-butylphenoxy) ethyl) thiourea).

[0036] Depend on Figure 2 NMR detection results of fluorescent probe TTBP-3C Link-FITC and Figure 4 The MS detection results of the fluorescent probe TTBP-3C Link-FITC indicate that the compound structure of the synthesized molecule is consistent with the expected probe molecular structure. Figure 3 The HPLC test results of the fluorescent probe TTBP-3C Link-FITC showed that the purity of the synthesized probe was higher than 95%.

[0037] Example 2 The binding constant of the specific fluorescent probe TTBP-3C Link-FITC to RXRα was detected. The specific steps are as follows: In this example, the RXRα protein was synthesized by OriGene Technologies, and its amino acid sequence is shown in the amino acid sequence table. It is a human RXRα ligand binding domain (RXRα-LBD) with a histidine tag connected to the C-terminus.

[0038] Binding experiments of specific fluorescent probes to different concentrations of RXRα protein were conducted to verify the activity of RXRα protein and select appropriate RXRα protein concentrations for subsequent experiments on the binding ability of compounds to RXRα; a TTBP-3CLink-FITC probe concentration of 20nM was selected; and final concentrations of RXRα protein concentrations of 0nM, 50nM, 100nM, 200nM, 400nM, 800nM, 1600nM, and 3200nM were selected.

[0039] (1) Prepare 2× protein concentrations: 0nM, 100nM, 200nM, 400nM, 800nM, 1600nM, 3200nM, and 6400nM; make three replicate wells, and prepare at least 35μL of each protein concentration.

[0040] (2) Prepare 2× concentration probe: 40 nM TTBP-3C Link-FITC probe.

[0041] (3) The volume of each well of the 384-well plate is 20µL, and 10µL protein + 10µL probe needs to be added; take 7 200µL centrifuge tubes for incubation of different concentrations of RXRα protein with 20nM TTBP-3C Link-FITC, use a spray gun to take 35µL of protein of each concentration + 35µL 100nM TTBP-3C Link-FITC and mix them in the 384-well plate, mix them in a 200µL centrifuge tube, incubate for 3-5 minutes, and then perform fluorescence polarization detection.

[0042] (4) 20 μL of the incubated solution was taken from the 200 μL centrifuge tube and added to the 384-well plate using an 8-channel dispenser; a SpectraMax i3x multifunctional microplate reader was used for detection.

[0043] The results showed that, with the increase of RXRα protein concentration, more probes were combined with RXRα protein, so the fluorescence polarization value showed a gradual upward trend; the fluorescence polarization value was plotted against the concentration of specific fluorescent probe TTBP-3C Link-FITC to obtain the binding curve; the Origin software was used to perform Sigmoidal fitting on the binding curve, and the GraphPad software was used to calculate the binding constant of specific fluorescent probe TTBP-3C Link-FITC and RXRα protein, which was 248.9 nmol / L. Figure 5

[0044] Example 3 The specific fluorescent probe was used to detect the binding ability of compound LG100268 and RXRα, and the specific steps were as follows: In this embodiment, RXRα activator LG100268 was a product of Med Chem Express Company (item number HY-15340), which was the test compound of Example 3, and dimethyl sulfoxide (DMSO) was used to prepare the mother solution; LG100268 was the ligand molecule of RXRα, which was a positive compound for the feasibility of the detection method; RXRα protein was synthesized by OriGene Technologies Company, and its amino acid sequence was shown in the amino acid sequence table, which was the ligand binding domain (RXRα-LBD) of human RXRα connected with a histidine tag at the C terminal.

[0045] Eight 200 μL EP tubes were taken, 35 μL of LG100268, 35 μL of specific fluorescent probe TTBP-3C Link-FITC and RXRα protein mixture prepared with potassium phosphate buffer were added to each tube, and the mixed samples were added to the 384-well plate at 20 μL per well using an 8-channel dispenser to obtain the detection system; in the detection system, the concentration of RXRα protein was 100 nmol / L, the concentration of specific fluorescent probe TTBP-3C Link-FITC was 20 nmol / L, and the concentration of LG100268 was 0 μmol / L, 0.15 μmol / L, 0.31 μmol / L, 0.62 μmol / L, 1.25 μmol / L, 2.5 μmol / L, 5 μmol / L and 10 μmol / L (three replicate wells were set for each LG100268 concentration); after the detection system was incubated at room temperature for 5 min, the fluorescence polarization of the FITC channel of the sample was detected by a multifunctional microplate reader, and the obtained fluorescence polarization average value was plotted against the concentration of LG100268 for statistical analysis. ​

[0046] The competition curve was fitted by Sigmoidal using Origin software, and the IC50 value of LG100268 was obtained 50 concentration value; By Figure 6 It can be seen that the fluorescence polarization signal of the system containing the test compound LG100268 is significantly lower than that of the 100% binding system (specifically, when no test compound is added, all fluorescent probes bind to the protein, which is defined as a 100% binding system). It can be judged that the test compound LG100268 can bind to RXRa, and the IC50 value thereof is obtained 50 concentration value (2.9 μmol / L).

[0047] Example 4 The binding ability of the compound tetrabromobisphenol A (TBBPA) to RXRa was detected by using specific fluorescent probes, and the specific steps were as follows: In this example, TBBPA is a product of Mreda / Merida Company (item number M057187), which is the test compound in Example 4, and dimethyl sulfoxide (DMSO) is used to prepare the mother solution; RXRa protein is synthesized by OriGene Technologies Company, and its amino acid sequence is shown in the amino acid sequence table. It is a human RXRa ligand binding domain (RXRa-LBD) with a histidine tag connected at the C-terminus.

[0048] 8 200 μL EP tubes were taken, 35 μL TBBPA, 35 μL specific fluorescent probe TTBP-3C Link-FITC prepared with potassium phosphate buffer and RXRa protein mixture were added to each tube, and the mixed sample was added to a 384-well plate with 8-channel gun at 20 μL per well to obtain a detection system. In the detection system, the concentration of RXRa protein is 100 nmol / L, the concentration of specific fluorescent probe TTBP-3C Link-FITC is 20 nmol / L, and the concentration of TBBPA is 0 μmol / L, 1.5 μmol / L, 3.1 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 25 μmol / L, 50 μmol / L and 100 μmol / L (3 replicates for each TBBPA concentration). After incubation at room temperature for 5 min, the fluorescence polarization of the sample FITC channel was detected by using a multifunctional enzyme label instrument, and the average fluorescence polarization value obtained was plotted against the TBBPA concentration for statistical analysis.

[0049] The competition curve was fitted by Sigmoidal using Origin software, and the IC50 value of TBBPA was obtained 50 concentration value; By Figure 7It can be seen that the fluorescence polarization signal of the system containing the test compound TBBPA in this embodiment is significantly lower than that of the 100% binding system, which means that the test compound TBBPA can bind to RXRα and its IC 50 Concentration value (45.5μmol / L).

[0050] By comparing the IC of LG100268 and TBBPA 50 value, IC of TBBPA 50 The IC value (45.5 μmol / L) of LG100268 was 50 The value (2.9 μmol / L) is 15 times higher, which indicates that the binding ability of TBBPA to RXRα is 1 / 15 of that of LG100268.

[0051] While various embodiments of the present invention have been described above, the foregoing description is intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to existing technologies, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A specific fluorescent probe, characterized in that: The specific fluorescent probe is a compound represented by formula (I); Formula (I) R is an inorganic group or an organic group that generates a fluorescent detection signal.

2. The specific fluorescent probe according to claim 1, characterized in that: The specific fluorescent probe is a compound represented by formula (II); Formula (II).

3. Use of the specific fluorescent probe according to claim 1 in detecting the binding ability of a compound to retinoid X receptor α.

4. The application according to claim 3, characterized in that: The test compound, specific fluorescent probe and retinoic acid X receptor α protein were co-incubated and the fluorescence polarization value was detected. 50 The concentration value is used to determine the binding ability of the test compound to retinoid X receptor α.

5. The application according to claim 4, characterized in that: The retinoid X receptor α is the following protein (a1) or (a2) or (a3): (a1) The amino acid sequence is the protein of sequence 1 in the sequence listing; (a2) a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the protein shown in Sequence 1 in the sequence listing; (a3) A protein having the same function as the amino acid sequence shown in Sequence 1 in the sequence listing, obtained by substituting and / or deleting and / or adding one or more amino acid residues.

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