A series of cGMP G-Flig probes, their applications and kits

Abstract

The application relates to the field of biomedical technology, and particularly relates to a cGMP G-Flig series probe. The G-Flig series comprises G-Flig1h, G-Flig1m, G-Flig1la and G-Flig1lb; the amino acid sequence of G-Flig1h is shown as SEQ ID NO:1; the amino acid sequence of G-Flig1m is shown as SEQ ID NO:2; the amino acid sequence of G-Flig1la is shown as SEQ ID NO:3; and the amino acid sequence of G-Flig1lb is shown as SEQ ID NO:4. The application has the following advantages: (1) a larger dynamic range; (2) high detection sensitivity; (3) high affinity, suitable for low-concentration cGMP cells; and (4) capable of being used in cGMP signal detection of active objects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a cGMP G-Flig series probe. Background Technology

[0002] Cyclic guanosine monophosphate (cGMP) is an important second messenger molecule in mammalian cells. Cellular and in vivo cGMP fluorescence imaging is a crucial direction for basic research on related signaling pathways and drug development. cGMP fluorescent probes are mainly divided into fluorescence resonance energy transfer (FRET) probes based on fluorescent proteins and single-fluoroprotein (SFP) based probes. The latter has a larger dynamic range and is simpler to use. Currently, all single-fluoroprotein-based cGMP probes are green, mainly including FlincG1, FlincG3, and Green cGull. However, these three probes show low brightness in cells cultured at 37°C, and FlincG1 and FlincG3 have very small dynamic ranges (ΔF / F0 < 100%).

[0003] cGMP fluorescence imaging in live cells refers to the expression of cGMP fluorescent probes in cells and then detecting changes in probe fluorescence intensity using fluorescence microscopy. Fluorescent probes are crucial for cGMP fluorescence imaging analysis. In recent years, internationally renowned laboratories have developed green cGMP cpFP probes with varying performance using different fluorescent proteins and cGMP sensing modules (Table 1), including the FlincG1 probe developed by Dr. Wolfgang R. Dostmann, the FlincG3 probe optimized by Dr. John Garthwaite, and the Green cGull probe developed by Dr. Tetsuya Kitaguchi.

[0004] However, the above probes cannot adequately meet the needs of live cell and live in vivo imaging, mainly due to the following deficiencies in key performance aspects (brightness, dynamic range, and affinity): (1) Small dynamic range in cells cultured at 37°C, such as FlincG1 and FlincG3 probes (Table 1). (2) Low fluorescence brightness in cells cultured at 37°C, such as FlincG1 and Green cGull probes (Table 1). (3) Flinc G3 and Green cGull have low affinity for cGMP (μM level), making them unsuitable for cells with low cGMP concentrations (such as adult cardiomyocytes and some neurons).

[0005] Table 1 Green cGMP probes based on a single fluorescent protein

[0006] Therefore, developing high-performance green cGMP probes and improving their brightness and dynamic range in practical applications is of great significance for enhancing detection sensitivity. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned technical problems and provide a cGMP G-Flig series probe with high fluorescence brightness, large dynamic range, and high detection sensitivity, which can detect changes in cGMP concentration after cell stimulation, as well as its applications and kits.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A cGMP G-Flig series probe, wherein the G-Flig series includes G-Flig1h, G-Flig1m, G-Flig11a, and G-Flig1lb; The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:4.

[0009] Furthermore, the excitation wavelength of the G-Flig series single photons is 470~500nm.

[0010] The present invention also provides a method for preparing cGMP G-Flig series probes.

[0011] A method for preparing cGMP G-Flig series probes, the method is as follows: using the GAF-A domain of mPDE5 that can bind cGMP as the sensing domain, linking it with the cyclized rearranged green fluorescent protein GFP, and after several amino acid mutations, the G-Flig1h, G-Flig1m, G-Flig1a, and G-Flig1lb probes are obtained. The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:4.

[0012] The present invention also provides a method for detecting cGMP fluorescence imaging in live cells.

[0013] A method for detecting cGMP fluorescence imaging in live cells, the method comprising the following steps: (1) Live cell culture, when the cell density is 60±5%, transfect plasmids containing G-Flig series probes; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 470~500nm for single photons.

[0014] The present invention also provides a method for detecting cGMP fluorescence imaging in mammalian cells.

[0015] A method for detecting cGMP fluorescence imaging in mammalian cells, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, plasmids containing G-Flig series probes were transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 470~500nm.

[0016] The present invention also provides another method for cGMP fluorescence imaging detection in mammalian cells.

[0017] A method for detecting cGMP fluorescence imaging in mammalian cells, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, plasmids containing G-Flig series probes were transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) The excitation wavelength of single photons is 470~500 nm. Fluorescence microscopy imaging analysis is performed at a frequency of 15 seconds per frame. 1 mM SNP is added in the tenth frame to detect the intensity change of G-Flig series probe fluorescence.

[0018] This invention also provides an application of the cGMP G-Flig series probes.

[0019] Application of a cGMP G-Flig series probe in cGMP detection.

[0020] This invention also provides another application of the cGMP G-Flig series probes.

[0021] Application of a cGMP G-Flig series probe in the detection of cGMP in live cells and / or live animals.

[0022] The present invention also provides a kit for G-Flig series probes.

[0023] A kit containing G-Flig series probes.

[0024] The cGMP G-Flig series probes provided by this invention use the GAF-A domain of mPDE5, which can bind to cGMP, as the sensing domain (sensing module), and interact with circularly permuted green fluorescent protein (GFP, cpGFP) via... Figure 1 The probes G-Flig1h, G-Flig1m, G-Fligl1a, and G-Flig1lb were obtained by connecting them in the manner shown and undergoing several amino acid mutations. Their sequences are shown in SEQ ID NO:1 to SEQ ID NO:4.

[0025] The cGMP G-Flig series probes provided by this invention insert circularly permuted green fluorescent protein (cpGFP) into a specific site of GAF-A to obtain a new cGMP fluorescent probe series G-Flig.

[0026] The cGMP G-Flig series probes provided by this invention are green cGMP probes. When cells are cultured at a physiological temperature of 37°C and under single-photon excitation (470~500nm), they exhibit high brightness (490%, 749%, 338%, and 368% of the brightest probe published so far, respectively) and a large dynamic range (ΔF / F0 are 267%, 223%, 330%, and 368%, respectively).

[0027] The cGMP G-Flig series probes provided by this invention have high affinity and are suitable for cells with low cGMP concentrations.

[0028] The core of this invention lies in the discovery of a green cGMP probe with higher brightness and a wider dynamic range. In practical applications, when expressed in mammalian cells, changes in cGMP concentration after specific stimulation can be detected using a conventional fluorescence microscope or two-photon microscope.

[0029] Compared with existing technologies, the advantages of the cGMP G-Flig series probes, their applications, and kits provided by this invention are as follows: (1) Larger dynamic range.

[0030] (2) High detection sensitivity.

[0031] (3) It has high affinity and is suitable for cells with low concentration of cGMP.

[0032] (4) It can be used in the detection of cGMP signals of moving objects. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the composition of the G-Flig series probes provided by the present invention; wherein, His is a protein purification tag, GAF (154~181) and GAFa (189~301) are the N-terminus and C-terminus of the sensing module, cpGFP is a cyclized rearranged green fluorescent protein (fluorescent module), and linker peptide 1 and linker peptide 2 are the connection parts between the sensing module and the fluorescent module. Figure 2 The present invention provides fluorescence excitation and emission spectra of the G-Flig series probes before and after cGMP binding. The bacteria expressing the probe are suspended in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH 7.2, sonicated and centrifuged, and the supernatant is removed to test the excitation and emission spectra of the probe before and after cGMP binding. The dashed line is the excitation spectrum and the solid line is the emission spectrum. Figure 3 This invention compares the fluorescence brightness of the G-Flig series probes (G-Flig1h, G-Flig1m, G-Flig1a, G-Flig1lb) and FlincG1, FlincG3, Green cGull, and GCaMP6s in Hek293T cells; among them, the commonly used green calcium ion probe GcaMP6s is used as a reference for fluorescence brightness. Figure 4The results show the response of different probes in HEK293T cells under single-photon excitation; (A) is a representative fluorescence image and response curve of the high-affinity probe G-Flig1h under 1mM SNP stimulation, and (B) is the maximum response amplitude of different probes (excitation wavelength 480nm±15nm, emission wavelength 530±15nm). The curve data are expressed as mean ± standard deviation (SD). Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions of the present invention, the following embodiments provide a more detailed description of the present invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0035] A cGMP G-Flig series probe, wherein the G-Flig series includes G-Flig1h, G-Flig1m, G-Flig11a, and G-Flig1lb; The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:4.

[0036] Furthermore, the excitation wavelength of the G-Flig series single photons is 470~500nm.

[0037] The present invention also provides a method for preparing cGMP G-Flig series probes.

[0038] A method for preparing cGMP G-Flig series probes, the method is as follows: using the GAF-A domain of mPDE5 that can bind cGMP as the sensing domain, linking it with the cyclized rearranged green fluorescent protein GFP, and after several amino acid mutations, the G-Flig1h, G-Flig1m, G-Flig1a, and G-Flig1lb probes are obtained. The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:4.

[0039] The present invention also provides a method for detecting cGMP fluorescence imaging in live cells.

[0040] A method for detecting cGMP fluorescence imaging in live cells, the method comprising the following steps: (1) Live cell culture, when the cell density is 60±5%, transfect plasmids containing G-Flig series probes; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 470~500nm for single photons.

[0041] The present invention also provides a method for detecting cGMP fluorescence imaging in mammalian cells.

[0042] A method for detecting cGMP fluorescence imaging in mammalian cells, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%. When the cell density was 60±5%, the plasmids of G-Flig series probes were transfected using the Lipofectamine 2000 kit. (2) After transfecting mammalian cells, culture them overnight, starve them for 2-4 hours with serum-free and phenol red-free medium, and then replace the medium with colorless and transparent live cell fluorescence imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 470~500nm.

[0043] The present invention also provides another method for cGMP fluorescence imaging detection in mammalian cells.

[0044] A method for detecting cGMP fluorescence imaging in mammalian cells, the method comprising the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%. When the cell density was 60±5%, the plasmids of G-Flig series probes were transfected using the Lipofectamine 2000 kit. (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) The excitation wavelength of single photons is 470~500nm. Fluorescence microscopy imaging analysis is performed at a frequency of one frame every 15 seconds. 1mM SNP is added in the tenth frame to detect the intensity change of G-Flig series probe fluorescence.

[0045] This invention also provides an application of the cGMP G-Flig series probes.

[0046] Application of a cGMP G-Flig series probe in cGMP detection.

[0047] This invention also provides another application of the cGMP G-Flig series probes.

[0048] Application of a cGMP G-Flig series probe in the detection of cGMP in live cells and / or live animals.

[0049] The present invention also provides a kit for G-Flig series probes.

[0050] A kit containing G-Flig series probes.

[0051] Example 1 Construction of G-Flig series probes Combination Figure 1 As shown, the GAF-A domain of mPDE5, which can bind cGMP, serves as the sensing domain (sensing module), and interacts with the circularly permuted green fluorescent protein GFP (cpGFP) via... Figure 1 The probes G-Flig1h, G-Flig1m, G-Fligl1a, and G-Flig1lb were obtained by connecting them in the manner shown and undergoing several amino acid mutations. Their sequences are shown in SEQ ID NO:1 to SEQ ID NO:4.

[0052] His is the protein purification tag; GAF (154-181) and GAFa (189-301) are the N-terminus and C-terminus of the sensing module, respectively. cpGFP is a cyclized rearranged green fluorescent protein (fluorescent module). Linker peptide 1 and linker peptide 2 are the connecting parts between the sensing module and the fluorescent module.

[0053] Example 2 Excitation and emission spectra of G-Flig series probes G-Flig1h, G-Flig1m, G-Fligl1a, and G-Flig1lb probes were expressed in bacteria. After culturing at room temperature for 3 days, bacterial cells were collected and sonicated in HEPES buffer (pH 7.2) containing 150 mM KCl and 50 mM HEPES. The supernatant (containing probes) was collected after centrifugation. 120 μL of the probe solution was added to 2 μL of HEPES solution or HEPES solution containing 30 mM cGMP. The response of the probes to a saturated concentration of cGMP (~500 μM) was detected using an Infinite M1000 PRO microplate reader. The results are shown below. Figure 2 As shown.

[0054] As shown in the figure, the signals of the G-Flig1h, G-Flig1m, G-Fligl1a, and G-Flig1lb probes increased by 6.8, 5.5, 8.8, and 8.4 times, respectively.

[0055] Example 3 Comparison of the brightness of G-Flig series probes (G-Flig1h, G-Flig1m, G-Fligl1a, G-Flig1lb) and FlincG1, FlincG3, Green cGull and GCaMP6s in Hek293T cells.

[0056] HEK293T cells were cultured in 12-well plates in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO2. When the cell density reached approximately 60%, the same mass of plasmids containing the expression probes G-Flig1h, G-Flig1m, G-Flig1a, G-Flig1lb, FlincG1, FlincG3, Green cGull, or GCaMP6s (the commonly used green calcium ion probe GCaMP6s was used as a fluorescence reference) were transfected into different wells using a Lipofectamine 2000 kit. After approximately 40 hours, the cells were washed with fluorescence imaging buffer, then resuspended in 200 μL of fluorescence imaging buffer and transferred to 96-well microplates. The plates were incubated at 37°C for 10 minutes, and finally, the fluorescence signal was detected using a microplate reader. Results are as follows: Figure 3 As shown.

[0057] Example 4 Response of different probes in HEK293T cells under single-photon excitation HEK293T cells should be cultured in glass-bottomed dishes in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C with 5% CO2. When the cell density is approximately 60%, plasmids containing the expression probes G-Flig1h, G-Flig1m, G-Fligl1a, G-Flig1lb, FlincG1, FlincG3, or Green cGull should be transfected into different dishes using a Lipofectamine 2000 kit.

[0058] After overnight culture, the cells were starved for 2 to 4 hours in serum- and phenol red-free medium (purchased from GIBCO). The medium was then replaced with colorless and transparent fluorescence imaging buffer, and imaging analysis was performed using an IX83 single-photon fluorescence microscope (excitation wavelength approximately 480 nm ± 15 nm, emission wavelength 530 ± 15 nm) built in our laboratory.

[0059] The imaging frequency was 15 s / scan. At the tenth frame, a final concentration of 1 mM SNP (NO donor, purchased from Beyotime Biotechnology Co., Ltd.) was added (e.g., if the culture dish contained 500 μL of fluorescence imaging buffer, 1.6 μL of 500 mM stock solution was dissolved in 300 μL of fluorescence imaging buffer; at the tenth frame, the diluted 300 μL SNP solution was added dropwise to the cell culture dish being imaged). Taking G-Flig 1h as an example, the change in fluorescence intensity in cells after 1 mM stimulation is shown in [the image / data]. Figure 4 .

[0060] A comparison of the maximum ΔF / F0 (peak ΔF / F0) for different probes can be found in [link to documentation]. Figure 4 This completes the fluorescence imaging step for changes in cGMP concentration within mammalian cells.

[0061] Figure 4 The curve data in the figure are represented as: mean ± standard deviation; ΔF / F0 is the ratio of the change in fluorescence intensity to the initial fluorescence intensity.

[0062] from Figure 4 As can be seen, HEK293T cells stimulated by SNPs exhibited a larger signal change amplitude (ΔF / F0) in G-Flig1h, with significant improvements in dynamic range and sensitivity.

[0063] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0064] It should also be noted that the various specific technical features and steps described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0065] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention. sequence list <110> Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences <120> A series of cGMP G-Flig probes, their applications and kits <130> 0 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 426 <212> PRT <213> Artificial sequence() <400> 1 Met Gly Ser His His His His His Gly Met Ala Ser Met Thr Gly 1 5 10 15 Gly Gly Gly Met Gly Ala Ala Leu Thr Ala Ala Ala Ala Leu Ala Pro 20 25 30 Gly Ser Leu Ala Val Thr Ala Leu Cys His Leu Ile Pro Leu His Ile 35 40 45 His Gly Leu Ile Ser Ala Ala Ala Thr Ser Leu Pro Leu Val Cys Thr 50 55 60 Pro Ala Val Thr Ile Thr Ala Ala Leu Gly Leu Ala Gly Ile Leu Ala 65 70 75 80 Ala Pro Leu Ile Gly His Ala Val Gly Gly Gly Gly Val Gly Leu Ala 85 90 95 Thr His Thr Gly Gly Ala Thr Pro Ile Gly Ala Gly Pro Val Leu Leu 100 105 110 Pro Ala Ala His Leu Leu Ser Val Gly Ser Leu Leu Ser Leu Ala Pro 115 120 125 Ala Gly Leu Ala Ala His Met Val Leu Leu Gly Pro Val Thr Ala Ala 130 135 140 Gly Ile Thr Pro Gly Met Ala Gly Leu Thr Leu Gly Gly Thr Gly Gly 145 150 155 160 Ser Met Val Ser Leu Gly Gly Gly Leu Pro Thr Gly Val Val Pro Val 165 170 175 Leu Val Gly Leu Ala Gly Ala Val Ala Gly His Leu Pro Ser Val Ala 180 185 190 Gly Gly Gly Gly Gly Ala Ala Thr Ala Gly Leu Leu Thr Leu Leu Pro 195 200 205 Ile Cys Thr Thr Gly Leu Leu Pro Val Pro Thr Pro Thr Leu Val Thr 210 215 220 Thr Leu Thr Thr Gly Val Gly Cys Pro Ala Ala Thr Pro Ala His Met 225 230 235 240 Leu Gly His Path Pro Pro Leu Ser Path Met Pro Gly Gly Thr Ile Gly 245 250 255 Gly Ala Thr Ile Val Pro Leu Ala Ala Gly Thr Thr Leu Thr Ala Ala 260 265 270 Gly Val Leu Pro Gly Gly Ala Thr Leu Val Ala Ala Ile Gly Leu Leu 275 280 285 Gly Ile Ala Pro Leu Gly Ala Gly Ala Ile Leu Gly His Leu Leu Gly 290 295 300 Thr Ala Thr Val Pro Leu Ile Ser Ala Leu Pro Ala Val Ala Gly Gly 305 310 315 320 Ser Thr Leu Gly Gly Ala Ser Ala Ala Cys Ile Ala Leu Gly Thr Ala 325 330 335 Leu Gly Ile Val Gly His Val Ala Ala Pro Gly Gly Pro Leu Ala Ile 340 345 350 Leu Ala Ala Thr Gly Ala Pro Ala Pro Ala Ala Gly Val Ala Gly Ile 355 360 365 Thr Gly Thr Leu Thr Gly Ser Ile Leu Cys Met Pro Ile Leu Ala His 370 375 380 Ala Gly Gly Val Val Gly Val Ala Gly Ala Ile Ala Leu Leu Ser Gly 385 390 395 400 Ala Gly Gly Thr Pro Thr Gly Leu Ala Gly Leu Ala Pro Ala Gly Thr 405 410 415 Leu Ala Thr Cys Gly Gly Val Leu His Ala 420 425 <210> 2 <211> 426 <212> PRT <213> Artificial Sequence() <400> 2 Met Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly 1 5 10 15 Gly Gly Gly Met Gly Ala Ala Leu Thr Ala Ala Ala Ala Leu Ala Pro 20 25 30 Gly Ser Leu Ala Val Thr Ala Leu Cys His Leu Ile Pro Leu His Ile 35 40 45 His Gly Leu Ile Ser Ala Ala Ala Thr Ser Leu Pro Leu Val Cys Thr 50 55 60 Pro Ala Val Thr Ile Thr Ala Ala Leu Gly Leu Ala Gly Ile Leu Ala 65 70 75 80 Ala Pro Leu Ile Ala His Ala Val Gly Gly Gly Gly Val Gly Leu Ala 85 90 95 Thr His Thr Gly Gly Ala Thr Pro Ile Gly Ala Gly Pro Val Leu Leu 100 105 110 Pro Ala Ala His Thr Leu Ser Val Gly Ser Leu Leu Ser Leu Ala Pro 115 120 125 Ala Gly Leu Ala Ala His Met Val Leu Leu Gly Pro Val Thr Ala Ala 130 135 140 Gly Ile Thr Gly Gly Met Ala Gly Leu Thr Leu Gly Gly Thr Gly Gly 145 150 155 160 Ser Met Val Ser Leu Gly Gly Gly Leu Pro Thr Gly Val Val Pro Val 165 170 175 Leu Val Gly Leu Ala Gly Ala Val Ala Gly His Leu Pro Ser Val Ala 180 185 190 Gly Gly Gly Gly Gly Ala Ala Thr Ala Gly Leu Leu Thr Leu Leu Pro 195 200 205 Ile Cys Thr Thr Gly Leu Leu Pro Val Pro Thr Pro Thr Leu Val Thr 210 215 220 Thr Leu Thr Thr Gly Val Gly Cys Pro Ala Ala Thr Pro Ala His Met 225 230 235 240 Leu Gly His Ala Pro Pro Leu Ser Ala Met Pro Gly Gly Thr Ile Gly 245 250 255 Gly Ala Thr Ile Val Pro Leu Ala Ala Gly Thr Thr Leu Thr Ala Ala 260 265 270 Gly Val Leu Pro Gly Gly Ala Thr Leu Val Ala Ala Ile Gly Leu Leu 275 280 285 Gly Ile Ala Pro Leu Gly Ala Gly Ala Ile Leu Gly His Leu Leu Gly 290 295 300 Thr Ala Thr Val Pro Leu Ile Ser Ala Leu Pro Ala Val Ala Gly Gly 305 310 315 320 Ser Thr Leu Gly Gly Ala Ser Ala Ala Cys Ile Ala Leu Gly Thr Ala 325 330 335 Leu Gly Ile Val Gly His Val Ala Ala Pro Gly Gly Pro Leu Ala Ile 340 345 350 Leu Ala Ala Thr Gly Ala Pro Ala Pro Ala Ala Gly Val Ala Gly Ile 355 360 365 Thr Gly Thr Leu Thr Gly Ser Ile Leu Cys Met Pro Ile Leu Ala His 370 375 380 Ala Gly Gly Val Val Gly Val Ala Gly Ala Ile Ala Leu Leu Ser Gly 385 390 395 400 Ala Gly Gly Thr Pro Thr Gly Leu Ala Gly Leu Ala Pro Ala Gly Thr 405 410 415 Leu Ala Thr Cys Gly Gly Val Leu His Ala 420 425 <210> 3 <211> 445 <212> PRT <213> artificial sequence() <400> 3 Met Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly 1 5 10 15 Gly Gly Gly Met Gly Ala Ala Leu Thr Ala Ala Ala Ala Leu Ala Pro 20 25 30 Gly Ser Leu Ala Val Thr Ala Leu Cys His Leu Ile Pro Leu His Ile 35 40 45 His Gly Leu Ile Ser Ala Ala Ala Thr Ser Leu Pro Leu Val Cys Thr 50 55 60 Pro Ala Val Thr Ile Thr Ala Ala Leu Gly Leu Ala Gly Ile Leu Ala 65 70 75 80 Ala Pro Leu Ile Gly His Ala Val Gly Gly Gly Gly Val Gly Leu Ala 85 90 95 Thr His Thr Gly Gly Ala Thr Pro Ile Gly Ala Gly Pro Val Leu Leu 100 105 110 Pro Ala Ala His Leu Leu Ser Val Gly Ser Leu Leu Ser Leu Ala Pro 115 120 125 Ala Gly Leu Ala Ala His Met Val Leu Leu Gly Pro Val Thr Ala Ala 130 135 140 Gly Ile Thr Leu Gly Met Ala Gly Leu Thr Leu Gly Gly Thr Gly Gly 145 150 155 160 Ser Met Val Ser Leu Gly Gly Gly Leu Pro Thr Gly Val Val Pro Val 165 170 175 Leu Val Gly Leu Ala Gly Ala Val Ala Gly His Leu Pro Ser Val Ala 180 185 190 Gly Gly Gly Gly Gly Ala Ala Thr Ala Gly Leu Leu Thr Leu Leu Pro 195 200 205 Ile Cys Thr Thr Gly Leu Leu Pro Val Pro Thr Pro Thr Leu Val Thr 210 215 220 Thr Leu Thr Thr Gly Val Gly Cys Pro Ala Ala Thr Pro Ala His Met 225 230 235 240 Leu Gly His Ala Pro Pro Leu Ser Ala Met Pro Gly Gly Thr Ile Gly 245 250 255 Gly Ala Thr Ile Val Pro Leu Ala Ala Gly Thr Thr Leu Thr Ala Ala 260 265 270 Gly Val Leu Pro Gly Gly Ala Thr Leu Val Ala Ala Ile Gly Leu Leu 275 280 285 Gly Ile Ala Pro Leu Gly Ala Gly Ala Ile Leu Gly His Leu Leu Gly 290 295 300 Thr Ala Thr Val Pro Leu Ile Ser Ala Leu Pro Ala Val Ala Gly Gly 305 310 315 320 Ser Thr Leu Gly Gly Ala Ser Ala Ala Cys Ile Ala Leu Gly Thr Ala 325 330 335 Leu Gly Ile Val Gly His Val Ala Ala Pro Gly Gly Pro Leu Ala Ile 340 345 350 Leu Ala Ala Thr Gly Ala Pro Ala Pro Ala Ala Gly Val Ala Gly Ile 355 360 365 Thr Gly Thr Leu Thr Gly Ser Ile Leu Cys Met Pro Ile Leu Ala His 370 375 380 Ala Gly Gly Val Val Gly Val Ala Gly Ala Ile Ala Leu Leu Ser Gly 385 390 395 400 Ala Gly Gly Thr Pro Thr Gly Leu Ala Gly Leu Ala Pro Ala Gly Thr 405 410 415 Leu Ala Thr Cys Gly Gly Val Leu Gly Ile Ala Gly Pro Cys Ala Thr 420 425 430 Pro Path Gly Thr Path Pro Leu Gly Ser Path Gly Pro Val 435 440 445 <210> 4 <211> 445 <212> PRT <213> artificial sequence() <400> 4 Met Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly 1 5 10 15 Gly Gly Gly Met Gly Ala Ala Leu Thr Ala Ala Ala Ala Leu Ala Pro 20 25 30 Gly Ser Leu Ala Val Thr Ala Leu Cys His Leu Ile Pro Leu His Ile 35 40 45 His Gly Leu Ile Ser Ala Ala Ala Thr Ser Leu Pro Leu Val Cys Thr 50 55 60 Pro Ala Val Thr Ile Thr Ala Ala Leu Gly Leu Ala Gly Ile Leu Ala 65 70 75 80 Ala Pro Leu Ile Gly His Ala Val Gly Gly Gly Gly Val Gly Leu Ala 85 90 95 Thr His Thr Gly Gly Ala Thr Pro Ile Gly Ala Gly Pro Val Leu Leu 100 105 110 Pro Ala Ala His Leu Leu Ser Val Gly Ser Leu Leu Ser Leu Ala Pro 115 120 125 Ala Gly Leu Ala Ala His Met Val Leu Leu Gly Pro Val Thr Ala Ala 130 135 140 Gly Ile Thr Ala Gly Met Ala Gly Leu Thr Leu Gly Gly Thr Gly Gly 145 150 155 160 Ser Met Val Ser Leu Gly Gly Gly Leu Pro Thr Gly Val Val Pro Val 165 170 175 Leu Val Gly Leu Ala Gly Ala Val Ala Gly His Leu Pro Ser Val Ala 180 185 190 Gly Gly Gly Gly Gly Ala Ala Thr Ala Gly Leu Leu Thr Leu Leu Pro 195 200 205 Ile Cys Thr Thr Gly Leu Leu Pro Val Pro Thr Pro Thr Leu Val Thr 210 215 220 Thr Leu Thr Thr Gly Val Gly Cys Pro Ala Ala Thr Pro Ala His Met 225 230 235 240 Leu Gly His Ala Pro Pro Leu Ser Ala Met Pro Gly Gly Thr Ile Gly 245 250 255 Gly Ala Thr Ile Val Pro Leu Ala Ala Gly Thr Thr Leu Thr Ala Ala 260 265 270 Gly Val Leu Pro Gly Gly Ala Thr Leu Val Ala Ala Ile Gly Leu Leu 275 280 285 Gly Ile Ala Pro Leu Gly Ala Gly Ala Ile Leu Gly His Leu Leu Gly 290 295 300 Thr Ala Thr Val Pro Leu Ile Ser Ala Leu Pro Ala Val Ala Gly Gly 305 310 315 320 Ser Thr Leu Gly Gly Ala Ser Ala Ala Cys Ile Ala Leu Gly Thr Ala 325 330 335 Leu Gly Ile Val Gly His Val Ala Ala Pro Gly Gly Pro Leu Ala Ile 340 345 350 Leu Ala Ala Thr Gly Ala Pro Ala Pro Ala Ala Gly Val Ala Gly Ile 355 360 365 Thr Gly Thr Leu Thr Gly Ser Ile Leu Cys Met Pro Ile Leu Ala His 370 375 380 Ala Gly Gly Val Val Gly Val Ala Gly Ala Ile Ala Leu Leu Ser Gly 385 390 395 400 Ala Gly Gly Thr Pro Thr Gly Leu Ala Gly Leu Ala Pro Ala Gly Thr 405 410 415 Leu Ala Thr Cys Gly Gly Val Leu His Ile Ala Gly Pro Cys Ala Thr 420 425 430 Pro Ala Gly Thr Ala Pro Leu Gly Ser Ala Gly Pro Val 435 440 445

Claims

1. A cGMP G-Flig series probe, characterized in that: The G-Flig series probes are G-Flig1h, G-Flig1m, G-Flig11a, or G-Flig1lb; The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:

4.

2. A method for preparing cGMP G-Flig series probes, characterized in that, The method is as follows: using the GAF-A domain of mPDE5, which can bind cGMP, as the sensing domain, and linking it to the cyclized rearranged green fluorescent protein GFP. After several amino acid mutations, the cGMP G-Flig series probes are obtained. The G-Flig series probes are G-Flig1h, G-Flig1m, G-Flig11a, or G-Flig1lb probes. The amino acid sequence of G-Flig1h is shown in SEQ ID NO:1; The amino acid sequence of G-Flig1m is shown in SEQ ID NO:2; The amino acid sequence of G-Fligl1a is shown in SEQ ID NO:3; The amino acid sequence of G-Flig1lb is shown in SEQ ID NO:

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3. The method for detecting cGMP fluorescence imaging in live cells for non-disease diagnosis or treatment purposes using the cGMP G-Flig series probes as described in claim 1, characterized in that, The method includes the following steps: (1) Live cell culture, when the cell density is 60±5%, transfect plasmids containing G-Flig series probes; (2) After transfecting, the cells were cultured overnight and starved for 2-4 hours. The culture medium was then replaced with a colorless and transparent buffer solution. (3) Fluorescence microscopy imaging analysis at excitation wavelengths of 470~500 nm for single photons.

4. The method for detecting cGMP fluorescence imaging in mammalian cells for non-disease diagnosis or treatment purposes using the cGMP G-Flig series probes as described in claim 1, characterized in that... The method includes the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, plasmids containing G-Flig series probes were transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) Fluorescence microscopy imaging analysis, in which the excitation wavelength of single photons is 470~500nm.

5. The method for detecting cGMP fluorescence imaging in mammalian cells for non-disease diagnosis or treatment purposes using the cGMP G-Flig series probes as described in claim 1, characterized in that... The method includes the following steps: (1) Mammalian cell culture: The culture medium was DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature was 37℃, and the CO2 content was 5%; When the cell density was 60±5%, plasmids containing G-Flig series probes were transfected using the Lipofectamine 2000 kit; (2) After transfecting mammalian cells, the cells were cultured overnight and starved for 2-4 hours in a medium without serum and phenol red. Then the medium was replaced with a colorless and transparent fluorescent imaging buffer. (3) The excitation wavelength of single photons is 470~500nm. Fluorescence microscopy imaging analysis is performed at a frequency of one frame every 15 seconds. 1mM SNP (Sodium Nitroprusside, NO donor) is added in the tenth frame to detect the intensity change of fluorescence of G-Flig series probes.

6. The application of the cGMP G-Flig series probes as described in claim 1 in the detection of cGMP for non-disease diagnosis or treatment purposes.

7. The application of the cGMP G-Flig series probes of claim 1 in the detection of cGMP in live cells and / or live animals for purposes other than disease diagnosis or treatment.

8. A kit comprising the G-Flig series probes as described in claim 1.

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