CN113567402B - Application of cAMP fluorescent probe G-Flamp1 - Google Patents
Application of cAMP fluorescent probe G-Flamp1 Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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Abstract
The invention discloses an application of a cAMP fluorescent probe G-Flamp1, wherein the cAMP fluorescent probe G-Flamp1 has single photon with excitation wavelength of 430-470nm or excitation wavelength of 880-920nm, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO: 1. Compared with the dynamic range of the existing fluorescent probe, the invention adopts 430-470nm single photon or 880-920nm two photon excitation G-Flamp1 probe to obtain the maximum dynamic range delta F/F in the cell culture at 37 DEG C 0 10-15, the detection sensitivity is greatly improved; in vivo brain slice neurons, ΔF/F under 60 μM Forskolin stimulation 0 The average value reaches 7, so that the expression and response in neurons can be very good; in practical use, G-Flamp1 is expressed in mammalian cells or living cells cultured ex vivo, and whether the cAMP concentration of the cells is changed after a specific stimulus is detected by using a fluorescence microscope or a two-photon microscope.
Description
Technical Field
The invention belongs to the technical field of biomedical optics and molecular imaging, and particularly relates to application of a cAMP fluorescent probe G-Flamp 1.
Background
Adenosine cyclophosphate (cAMP) is currently the largest drug target downstream messenger molecule of the G Protein Coupled Receptor (GPCR) family, and cAMP fluorescent probe and its live cell and living body microscopic imaging are important directions for fundamental research and drug development of GPCR signaling pathways. cAMP fluorescence imaging in living cells refers to the expression of cAMP fluorescent probes in cells and then detecting changes in the probe fluorescent signal (e.g. intensity) using a fluorescence microscope. Fluorescent probes are key to cAMP fluorescence imaging analysis. The cAMP fluorescent probes are mainly classified into fluorescent protein-based fluorescence resonance energy transfer probes and single fluorescent protein-based probes, and the latter probes have a larger dynamic range than the former probes and are simple to use. At present, cAMP probes based on a single fluorescent protein are classified into green and Red 2 subclasses, the former mainly comprising Flamindo2, cADDIS and cAMPr, and the latter mainly comprising Pink Flamindo, red cADDIS and R-FlincA. The dynamic range of the probe was small in cells cultured at 37℃physiological temperature, as shown in the following table. In practical application, dynamic range (amplitude of change in fluorescence brightness, ΔF/F 0 ) Is an important parameter and is directly related to the detection sensitivity. Therefore, the dynamic range of the probe in practical application is improved, and the method has important significance for improving the detection sensitivity.
Reference is made to:
1.Odaka H,Arai S,Inoue T,Kitaguchi T(2014)Genetically-encoded yellow fluorescent cAMP indicator with an expanded dynamic range for dual-color imaging.PLoS One 9:e100252.
2.Tewson PH,Martinka S,Shaner NC,Hughes TE,Quinn AM(2016)New DAG and cAMP Sensors Optimized for Live-Cell Assays in Automated Laboratories.J Biomol Screen 21:298-305.
3.Hackley CR,Mazzoni EO,Blau J(2018)cAMPr:A single-wavelength fluorescent sensor for cyclic AMP.Sci Signal 11.
CN1096270444A, 2019.4.16, cAMP fluorescent probe and application thereof
Adenosine cyclophosphate fluorescent probe with wide variation of fluorescent brightness in CN201911251920.X
6.Harada K,Ito M,Wang X,Tanaka M,Wongso D,et al.(2017)Red fluorescent protein-based cAMP indicator applicable to optogenetics and in vivo imaging.Sci Rep 7:7351
7.https://montanamolecular.com/live-cell-camp-assay-caddis/red-caddis-camp-protocol/
8.Ohta Y,Furuta T,Nagai T,Horikawa K(2018)Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.Sci Rep 8:1866.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention aims to provide an application of a cAMP fluorescent probe G-Flamp 1.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of a cAMP fluorescent probe G-Flamp1 in single photon imaging, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1, wherein the excitation wavelength of the single photon is 430-470nm.
Further, the excitation wavelength of the single photon is 430-450nm; preferably, the excitation wavelength of the single photon is 450nm.
The invention also provides an application of the cAMP fluorescent probe G-Flamp1 in two-photon imaging, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1, wherein the excitation wavelength of the two photons is 880-920nm.
Further, the excitation wavelength of the two photons is 900-920nm; preferably, the excitation wavelength of the two photons is 900nm and 920nm.
The invention also provides an application of the cAMP fluorescent probe G-Flamp1 in cAMP signal detection, wherein the excitation wavelength of single photon adopted in the signal detection is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
In a further aspect, the invention provides an application of a cAMP fluorescent probe G-Flamp1 in detecting cAMP signals in living cells, wherein the excitation wavelength of single photons adopted in the signal detection is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
In a further aspect, the present invention provides a method for detecting cAMP fluorescence imaging in living cells, comprising the steps of:
1) Expressing cAMP fluorescent probe G-Flamp1 in mammalian cells;
2) Performing imaging analysis by using a fluorescence microscope or a two-photon microscope, and detecting the intensity change of probe fluorescence, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm;
preferably, the method comprises the following steps: 1) Expressing cAMP fluorescent probe G-Flamp1 in mammalian cells;
2) Stimulating mammalian cells to increase or decrease their intracellular cAMP concentration;
3) And (3) performing imaging analysis by using a fluorescence microscope or a two-photon microscope, and detecting the intensity change of the fluorescence of the probe before and after the stimulation, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
In a further aspect, the invention provides an application of a cAMP fluorescent probe G-Flamp1 in-vivo brain-in-tablet cAMP signal detection, wherein the excitation wavelength of single photon adopted in the signal detection is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
In a further aspect, the present invention provides a method for detecting cAMP fluorescence imaging in a brain slice of a living body, comprising the steps of:
1) Injecting a viral vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) After the G-Flamp1 probe is expressed in brain neurons, preparing a living brain slice;
3) Performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscopic endoscope, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm;
preferably, the method comprises the following steps: 1) Injecting a viral vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) After the G-Flamp1 probe is expressed in brain neurons, preparing a living brain slice;
3) Stimulating neurons in the brain region of a mammal to increase or decrease their intracellular cAMP concentration;
4) Performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscopic endoscope; the excitation wavelength of the single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of the two photons is 880-920nm.
In a further aspect, the invention provides an application of a cAMP fluorescent probe G-Flamp1 in detection of cAMP signals in living animals, wherein the excitation wavelength of single photons adopted in the detection of the signals is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
In a further aspect, the present invention provides a method for detecting cAMP fluorescence imaging in a living body, comprising the steps of:
1) Injecting a virus vector containing a G-Flamp1 probe gene into a target tissue organ of a mammal;
2) Performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscopic endoscope, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm;
preferably, the method comprises the following steps: 1) Injecting a virus vector containing a G-Flamp1 probe gene into a target tissue organ of a mammal;
2) Stimulating cells in a target tissue organ of a mammal to raise or lower their intracellular cAMP concentration;
3) Performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscopic endoscope; the excitation wavelength of the single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of the two photons is 880-920nm.
As is well known in the art, the fluorescence excitation wavelength is usually determined according to the excitation line of such luciferin, and the peak value is usually taken, and the peak value of the excitation spectrum of the G-Flamp1 probe adopted by the invention is about 495nm, so that single photons with the excitation wavelength of 480nm are adopted in the prior application CN201911251920.X, and the cAMP dynamic range (delta F/F) in cells is cultivated at 37 ℃ physiological temperature 0 ) 2.2, the application cultures cAMP dynamic range (DeltaF/F) in cells by using single photon with excitation wavelength of 430-470nm or two photon with excitation wavelength of 880-920nm at 37 ℃ physiological temperature 0 ) And the number is 10-15, unexpected technical effects are obtained, and the method is creative.
The beneficial effects of the invention are as follows: compared with the dynamic range of the existing fluorescent probe, the invention adopts 430-470nm single photon or 880-920nm two photon excitation G-Flamp1 probe in the physiological temperature culture cell at 37 ℃ to obtain the current maximum dynamic range (delta F/F) 0 About 10 to 15, ratio delta F/F 0 2.2 is obviously high), and the detection sensitivity is greatly improved; in vivo brain slice neurons, ΔF/F under 60 μM Forskolin stimulation 0 The average value reaches 7, so that the expression and response in neurons can be very good; in practical use, G-Flamp1 is expressed in mammalian cells or living cells cultured in vitro, and whether the cAMP concentration of the cells is changed after specific stimulation can be detected by using a common fluorescence microscope or a two-photon microscope.
Drawings
FIG. 1 shows the amino acid sequence # 252 and the G-Flamp1 sequence in example 1 of the present invention.
FIG. 2 is an excitation and emission spectrum of a purified G-Flamp1 probe in example 2 of the present invention, the purified G-Flamp1 probe from bacteria was diluted in HEPES solution at pH 7.3 to a final concentration of 2. Mu.M, and the fluorescence excitation spectrum of the probe in HEPES solution and saturated concentration of cAMP was shown, with a dotted line being the excitation spectrum, a solid line being the emission spectrum, and 500. Mu.M cAMP (bold line): the probe solution contained cAMP, HEPES buffer (thin line): the probe was in HEPES buffer without cAMP.
FIG. 3 (A) shows the dynamic range measurement of the purified G-Flamp1 probe of example 3 of the present invention under single photon excitation at different excitation wavelengths, FIG. 3 (B) shows the dynamic range measurement of the purified G-Flamp1 probe of example 3 of the present invention under two photon excitation at different excitation wavelengths, the purified G-Flamp1 probe from bacteria was diluted in HEPES solution at pH 7.3 to a final concentration of 2. Mu.M, showing the fluorescence excitation spectrum ratio of the probe at saturated concentration of cAMP and without cAMP, F 0 The fluorescence intensities of the probe in the presence of saturated concentration of cAMP (500. Mu.M) and in the absence of cAMP, respectively.
FIG. 4 is a graph showing normalized signal change amplitude measurement of the purified G-Flamp1 probe at various cAMP concentrations in example 4 of the present invention, wherein the left graph shows a test chart under excitation at 480nm wavelength, and the right graph shows a test chart under excitation at 450nm wavelength.
FIG. 5 is a graph showing the comparison of fluorescence intensity of different probes in HEK293T cells according to example 5 of the present invention.
FIG. 6 is a graph showing the response of different probes in HEK293T cells under single photon excitation in example 6 of the present invention; (A) HEK293T cell plasmids containing cAMPr, flamindo, R-FlincA and Pink-Flamindo probes were transfected with Lipofectamine, respectively, and after overnight culture, the HEK293T cell plasmids were starved with DMEM cell culture solution containing no phenol red and serum for 6 hours, and after stimulation with 60. Mu.M Forskolin (Fsk), the fluorescence brightness was changed; (B) response of G-Flamp1 probe; G-Flamp1 excitation wavelength is 440+ -10 nm, fluorescence receiving wavelength is 530+ -15 nm, cAMPr and Flamindo2 excitation wavelength is 480+ -15 nm, fluorescence receiving wavelength is 530+ -15 nm, R-FlincA and Pink-Flamindo excitation wavelength is 568+ -10 nm, and fluorescence receiving wavelength is 630+ -25 nm; the curve data represents: mean ± standard deviation; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity.
FIG. 7 is a diagram of embodiment 9 of the present inventionResponse of G-Flamp1 probe in HEK293T cells under 20nm two-photon excitation; HEK293T cells transfected with plasmid containing G-Flamp1 probe by Lipofectamine, after overnight culture, after starving with DMEM cell culture solution without phenol red and serum for 6 hours, fluorescence brightness change after stimulation with 60. Mu.M Forskolin (Fsk); the two-photon excitation wavelength is 920nm; the different curves represent the fluorescence response of different cells; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity.
FIG. 8 is a response of a G-Flamp1 probe in HEK293T cells under 900nm two-photon excitation in example 8 of the present invention; HEK293T cells transfected with plasmid containing G-Flamp1 probe by Lipofectamine, after overnight culture, after starving with DMEM cell culture solution without phenol red and serum for 6 hours, fluorescence brightness change after stimulation with 60. Mu.M Forskolin (Fsk); the two-photon excitation wavelength is 900nm; the different curves represent the fluorescence response of different cells; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity.
FIG. 9 is a graph showing the results of two-photon fluorescence imaging of a living brain slice neuron according to example 9 of the present invention, wherein (A) is a graph of fluorescence intensity of cells before and after stimulation of the brain slice neuron by Fsk, and the scale is: 50 microns; (B) Is the change in fluorescence intensity (ΔF/F) of the representative neuronal cell bodies in (A) 0 ) Curve, deltaF/F 0 For the ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity, different gray curves were from different cells, and the black curve was the average of the gray curves.
Detailed Description
For a better understanding of the present invention, the following description will be given with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following examples.
Example 1#252 amino acid sequence and G-Flamp1 sequence
The amino acid sequences of #252 (the amino acid sequence is shown as SEQ ID NO: 2) are mutated to obtain the G-Flamp1 probe (the amino acid sequence is shown as SEQ ID NO: 1), and the amino acid sequences of #252 and G-Flamp1 are shown as figure 1.
Underlined amino acids are components of the tandem peptides, respectively. Between WG and RV is a circularized rearranged green fluorescent protein sequence (bolded). WG is preceded by the N-terminal sequence of msicnbd and RV is followed by the C-terminal sequence of msicnbd. The amino acids that are bolded and tilted are mutated amino acids.
EXAMPLE 2 purification of excitation and emission spectra of G-Flamp1 probes
G-Flamp1 probe was expressed in bacteria, cells were collected by culturing at room temperature for 2 days, sonicated in HEPES buffer (containing 150mM KCl and 50mM HEPES) at pH=7.3, the probe was purified by HisPur Cobalt Resin (available from Pierce Co.), and the probe was dissolved in HEPES buffer at pH=7.3 by Econo-Pac 10DG desalting column (available from Bio-Rad Co., U.S.A.), and the probe concentration was determined using BCA kit (available from Thermo scientific Co., U.S.A.). To 2 wells of a 96-well plate containing 120. Mu.L of 2. Mu.M probe solution, 2. Mu.L of HEPES buffer and 2. Mu.L of 30mM cAMP solution (final concentration 500. Mu.M) were added, respectively, and then probe excitation and emission spectra were detected using a multifunctional microplate reader Infinite M1000 PRO, as shown in FIG. 2. In the presence of saturated concentration cAMP (500. Mu.M), the maximum peak of the excitation and emission spectra of the purified G-Flamp1 probe corresponds to wavelengths of 495nm and 515nm, respectively; the maximum peak of the excitation and emission spectra of the purified G-Flamp1 probe in the HEPES buffer without cAMP corresponds to 502nm and 517nm, respectively. By comparing the peak size of the fluorescence emission spectra (peak 0.106 for the probe in HEPES solution and 1 at saturated cAMP concentration), it was seen that the probe fluorescence brightness increased 8.4 times in the presence of saturated concentration cAMP (500. Mu.M) compared to HEPES buffer.
Example 3 dynamic Range determination of purified G-Flamp1 Probe under Single photon and two photon excitation at different excitation wavelengths
G-Flamp1 probe was expressed in bacteria, cells were collected by culturing at room temperature for 2 days, sonicated in HEPES buffer (containing 150mM KCl and 50mM HEPES) at pH=7.3, the probe was purified by HisPur Cobalt Resin (available from Pierce Co.), and the probe was dissolved in HEPES buffer at pH=7.3 by Econo-Pac 10DG desalting column (available from Bio-Rad Co., U.S.A.), and the probe concentration was determined using BCA kit (available from Thermo scientific Co., U.S.A.). To 2 wells of a 96-well plate containing 120. Mu.L of a 2. Mu.M probe solution, 2. Mu.L of HEPES buffer and 2. Mu.L of 30mM cAMP solution (final concentration: 500. Mu.M) were added, respectively, and the ratio of fluorescence brightness of the probes containing saturated cAMP concentration to those of probes not containing cAMP under single-photon and two-photon excitation was as shown in FIG. 3, and it was found that the maximum dynamic ranges were around 450nm and 900nm, respectively.
Example 4 affinity of probes for cAMP
The purified probe (2. Mu.M concentration) of example 3 was mixed with cAMP at different concentrations, respectively, to obtain a dose-response curve. As shown in FIG. 4, the affinity of cAMP was about 2.3. Mu.M under excitation at 480nm and 450nm, which satisfies most of the application requirements.
Example 5 comparison of fluorescent brightness of different probes in HEK293T cells
The calcium ion probe GCaMP6s is a classical gene coding probe and is widely used for living cells and living body imaging, and the fluorescence brightness of the calcium ion probe GCaMP6s can be used as a reference of the gene coding probe. Several gene-encoded probes, such as calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamindo2/G-Flamp1, etc., were constructed separately on eukaryotic expression vectors (CAG promoter), and HEK293T cells (purchased from GE Healthcare Dharmacon company) cultured in glass-bottomed dishes were transfected by Lipofectamine 2000 kit. After culturing at 37℃for 48 hours, the cell suspension was collected into an imaging buffer, and the calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamindo2/G-Flamp1 fluorescence intensity under 480nm excitation and G-Flamp1 probe fluorescence intensity under 450nm excitation were detected by an enzyme-labeled instrument, and the detection results are shown in FIG. 5, wherein the relative fluorescence intensities of the calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamindo2/G-Flamp1 under 480nm excitation were 1, 0.33, 0.28, 0.47, and G-Flamp1 was higher than cAMPr/Flamindo2 probe, respectively, and were 44% of the widely used GCaMP6 s. The G-Flamp1 probe has a relative fluorescence intensity of 0.22 under the excitation of 450nm, and the fluorescence intensity is equivalent to that of other green cAMP probes.
Example 6 response of different probes in HEK293T cells under Single photon excitation
Probes such as cAMPr, flamindo, G-Flamp1, pink-Flamindo and R-FlincA were respectively constructed on eukaryotic expression vectors (CAG promoter), and HEK293T cells (purchased from GE H) cultured in glass-bottomed dishes were transfected with Lipofectamine 2000 kitealthcare Dharmacon company) and starved the cells for 6 hours with serum-free, phenol red-free medium (available from the company GIBCO). The brightness of the probe was detected by using an IX83 fluorescence microscope self-constructed in the laboratory, the G-Flamp1 excitation wavelength was 440.+ -.10 nm, the fluorescence receiving wavelength was 530.+ -.15 nm, the campr and Flamindo2 excitation wavelength was 480.+ -.15 nm, the fluorescence receiving wavelength was 530.+ -.15 nm, the R-FlincA and Pink-Flamindo excitation wavelength was 568.+ -.10 nm, the fluorescence receiving wavelength was 630.+ -.25 nm, and the signal change amplitude (. DELTA.F/F) of each probe after the visible cell was stimulated with 60. Mu.M Forskolin (purchased from Biyunshii Biotechnology Co.) 0 ) As shown in fig. 6. The fluorescence imaging step of the change in the intracellular cAMP concentration of the mammal is completed. The curve data in fig. 6 represents: mean ± standard deviation; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity. From FIG. 6 (A), it can be seen that the signal amplitude of cAMPr is 0.45, that of Flamindo2 is 0.26, that of Pink-Flamindo is 0.89, that of R-FlincA is 1.28, and that of G-Flamp1 is 10. As can be seen by comparison, G-Flamp1 has the greatest signal change amplitude (ΔF/F) after stimulation of 60. Mu.M Forskolin (available from Biyun Biotechnology Co.) 0 ) The dynamic range and the sensitivity are greatly improved.
Example 7 response of G-Flamp1 Probe in HEK293T cells under two-photon excitation at 920nm
G-Flamp1 probe was constructed on eukaryotic expression vector (CAG promoter), HEK293T cells (purchased from GE Healthcare Dharmacon company) cultured in glass-bottomed dishes were transfected by Lipofectamine 2000 kit, and after overnight culture, the cells were starved with serum-free, phenol red-free medium (purchased from GIBCO company) for 6 hours. Imaging analysis was performed using a commercial two-photon microscope with a two-photon excitation wavelength of 920nm, and the change in fluorescence intensity of G-Flamp1 in the various cells after stimulation of the visible cells with 60. Mu.M Forskolin (available from Biyun Biotechnology Co.) was shown in FIG. 7. Visible G-Flamp1 fluorescence intensity display in different cells after 60. Mu.M Forskolin (purchased from Biyun Biotechnology Co.) stimulationShowing different variations of ΔF/F 0 The average value is 10 (currently up to 2.2). Wherein the different curves represent the fluorescence response of different cells; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity.
Example 8 response of G-Flamp1 Probe in HEK293T cells under two-photon excitation at 900nm
As shown in FIG. 8, the fluorescence intensity of G-Flamp1 in different cells showed different changes, ΔF/F, by changing the excitation wavelength to 900nm only 0 The average value is 14 (currently up to 2.2). Wherein the different curves represent the fluorescence response of different cells; ΔF/F 0 The ratio of the amount of change in fluorescence intensity to the initial fluorescence intensity.
Example 9 two-photon fluorescence imaging of brain slice neurons
AAV virus containing the G-Flamp1 gene was injected into the mouse striatal neuronal region. After 4 weeks, mice were anesthetized and a living brain patch (300 μm thick) was prepared. The brain slice is incubated in artificial cerebrospinal fluid at 33 degrees celsius for 20-30 minutes and then at room temperature for 20-30 minutes. Finally, the change of fluorescence signals of striatal neurons before and after the stimulation of 60 mu M Forskolin is monitored by a two-photon fluorescence microscope system under a 25-fold water microscope and a 920nm excitation line. The results are shown in FIG. 9, which shows the fluorescence intensity of cells before and after Fsk stimulation on the scale of (A): from the figure, it can be seen that the fluorescence intensity of cells after Fsk stimulation is significantly enhanced at 50 μm. (B) Is the change in fluorescence intensity (ΔF/F) of the representative neuronal cell bodies in (A) 0 ) Curves, different grey curves from different cells, black curves being the mean of the grey curves, as can be seen from the figure, ΔF/F 0 The average value reaches 7, and the expression and response in neurons can be very good.
Example 10 Living body detection
Injecting a viral vector containing a probe gene into a brain region of interest of a mouse; after 3-4 weeks, cortical neurons in the brain area of the mice were stimulated to increase or decrease their intracellular cAMP concentration and analyzed by imaging using two-photon fluorescence microscopy or microscopy. For tissue shallow cells, the signal change of the probe can also be detected by a single photon fluorescence microscope.
In summary, in the physiological temperature culture of cells at 37 ℃, 430-470nm single photon or 880-920nm two photon excitation G-Flamp1 probe is adopted, and the maximum dynamic range (delta F/F) is achieved 0 10-15), the increase of the dynamic range can improve the detection sensitivity; in vivo brain slice neurons, ΔF/F under 60 μM Forskolin stimulation 0 The average value reaches 7, so that the expression and response in neurons can be very good; in practical use, G-Flamp1 is expressed in mammalian cells or living cells cultured in vitro, and whether the cAMP concentration of the cells is changed after specific stimulation can be detected by using a common fluorescence microscope or a two-photon microscope.
The foregoing is merely a specific embodiment of the present invention and not all embodiments, and any equivalent modifications of the technical solution of the present invention that will be obvious to those skilled in the art from reading the present specification are intended to be encompassed by the claims of the present invention.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of China academy of sciences
<120> application of cAMP fluorescent probe G-Flamp1
<130> CP120010177C
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 422
<212> PRT
<213> artificial sequence
<400> 1
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Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365
Ile Ser Gly Glu Pro Arg Val Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415
Gly Ala Ala Ala Ser Ala
420
<210> 2
<211> 422
<212> PRT
<213> artificial sequence
<400> 2
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
1 5 10 15
Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
20 25 30
Pro Met Gly Phe Tyr Gln Glu Val Arg Arg Gly Asp Phe Val Arg Asn
35 40 45
Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
50 55 60
Val Leu Val Glu Ile Val Arg Ala Leu Arg Ala Arg Thr Val Pro Ala
65 70 75 80
Gly Ala Val Ile Cys Arg Ile Gly Glu Pro Gly Asp Arg Met Phe Phe
85 90 95
Val Val Glu Gly Ser Val Ser Val Ala Thr Pro Trp Gly Asn Val Tyr
100 105 110
Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
115 120 125
Arg His Asn Val Glu Asp Gly Gly Val Gln Leu Ala Tyr His Tyr Gln
130 135 140
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His
145 150 155 160
Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg
165 170 175
Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu
180 185 190
Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Met Val Ser
195 200 205
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
210 215 220
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu
225 230 235 240
Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr
245 250 255
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr
260 265 270
Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
275 280 285
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile
290 295 300
Val Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe
305 310 315 320
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
325 330 335
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Arg Val
340 345 350
Asn Pro Val Glu Leu Gly Pro Gly Ala Phe Phe Gly Glu Met Ala Leu
355 360 365
Ile Ser Gly Glu Pro Arg Ser Ala Thr Val Ser Ala Ala Thr Thr Val
370 375 380
Ser Leu Leu Ser Leu His Ser Ala Asp Phe Gln Met Leu Cys Ser Ser
385 390 395 400
Ser Pro Glu Ile Ala Glu Ile Phe Arg Lys Thr Ala Leu Glu Arg Arg
405 410 415
Gly Ala Ala Ala Ser Ala
420
Claims (15)
1. An application of a cAMP fluorescent probe G-Flamp1 in single photon imaging, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1, wherein the excitation wavelength of the single photon is 430-470nm.
2. The use according to claim 1, wherein the single photon has an excitation wavelength of 430-450 nm.
3. The use according to claim 2, wherein the single photon has an excitation wavelength of 450nm.
4. Application of a cAMP fluorescent probe G-Flamp1 in two-photon imaging, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1, wherein the excitation wavelength of the two photons is 880-920nm.
5. The use according to claim 4, wherein the excitation wavelength of the two photons is 900-920 nm.
6. The use according to claim 5, wherein the excitation wavelength of the two photons is 900nm, 920nm.
7. An application of a cAMP fluorescent probe G-Flamp1 in detecting cAMP signals in living cells, which is characterized in that the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1 is shown in the specification; the excitation wavelength of single photon adopted in the signal detection is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
8. A method for detecting cAMP fluorescence imaging in living cells, comprising the steps of:
1) Expressing a cAMP fluorescent probe G-Flamp1 in a mammalian cell, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1 is shown in the specification;
2) The method comprises the steps of performing imaging analysis by using a single photon fluorescence microscope or a two-photon microscope, and detecting the intensity change of probe fluorescence, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
9. The method for detecting cAMP fluorescence imaging in living cells according to claim 8, comprising the steps of:
1) Expressing a cAMP fluorescent probe G-Flamp1 in a mammalian cell, wherein the amino acid sequence of the G-Flamp1 is shown in SEQ ID NO:1 is shown in the specification;
2) Stimulating mammalian cells to increase or decrease their intracellular cAMP concentration;
3) The method comprises the steps of performing imaging analysis by using a single photon fluorescence microscope or a two-photon microscope, and detecting the intensity change of probe fluorescence, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
10. The application of a cAMP fluorescent probe G-Flamp1 for non-disease diagnosis and treatment purpose in-vivo cAMP signal detection in brain slices is characterized in that the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1, wherein the excitation wavelength of single photon adopted in the signal detection is 430-470nm; the excitation wavelength of the two photons is 880-920nm.
11. A method for detecting cAMP fluorescence imaging in a living brain slice for non-disease diagnosis and treatment purposes, comprising the steps of:
1) Injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal, wherein the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1 is shown in the specification;
2) After the G-Flamp1 probe is expressed in brain neurons, preparing a living brain slice;
3) Imaging analysis is carried out by using a single photon fluorescence microscope, a two photon fluorescence microscope or a microscopic endoscope, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
12. A method for fluorescent imaging detection of cAMP in a living brain slice for non-disease diagnosis and treatment purposes according to claim 11, comprising the steps of:
1) Injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal, wherein the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1 is shown in the specification;
2) After the G-Flamp1 probe is expressed in brain neurons, preparing a living brain slice;
3) Stimulating neurons in the brain region of a mammal to increase or decrease their intracellular cAMP concentration;
4) Performing imaging analysis by using a single photon fluorescence microscope, a two photon fluorescence microscope or a microscopic endoscope; the excitation wavelength of the single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of the two photons is 880-920nm.
13. An application of a cAMP fluorescent probe G-Flamp1 for non-disease diagnosis and treatment purpose in detection of cAMP signal in living animals, which is characterized in that the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1 is shown in the specification; the excitation wavelength of single photon adopted in the signal detection is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
14. A method for detecting cAMP fluorescence imaging in an active body for non-disease diagnosis and treatment purposes, comprising the steps of:
1) Injecting a virus vector containing a G-Flamp1 probe gene into a target tissue organ of a mammal, wherein the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1 is shown in the specification;
2) Imaging analysis is carried out by using a single photon fluorescence microscope, a two photon fluorescence microscope or a microscopic endoscope, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
15. A method for the fluorescence imaging detection of cAMP in a living body for non-disease diagnosis and treatment purposes according to claim 14, comprising the steps of:
1) Injecting a virus vector containing a G-Flamp1 probe gene into a target tissue organ of a mammal, wherein the amino acid sequence of the G-Flamp1 is shown as SEQ ID NO:1 is shown in the specification;
2) Stimulating cells in a target tissue organ of a mammal to raise or lower their intracellular cAMP concentration;
3) Imaging analysis is carried out by using a single photon fluorescence microscope, a two photon fluorescence microscope or a microscopic endoscope, wherein the excitation wavelength of single photons adopted in the imaging analysis is 430-470nm, and the excitation wavelength of two photons is 880-920nm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015066706A3 (en) * | 2013-11-04 | 2015-06-25 | Montana Molecular Llc | GENETICALLY ENCODED FLUORESCENT SENSORS FOR DETECTING LIGAND BIAS AND INTRACELLULAR SIGNALING THROUGH cAMP PATHWAYS |
| DE102015115640A1 (en) * | 2015-09-16 | 2017-03-16 | Stiftung Caesar Center Of Advanced European Studies And Research | Method for the detection of ligands by means of biosensors |
| CN109627344A (en) * | 2018-12-28 | 2019-04-16 | 深圳先进技术研究院 | CAMP fluorescence probe and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015066706A3 (en) * | 2013-11-04 | 2015-06-25 | Montana Molecular Llc | GENETICALLY ENCODED FLUORESCENT SENSORS FOR DETECTING LIGAND BIAS AND INTRACELLULAR SIGNALING THROUGH cAMP PATHWAYS |
| DE102015115640A1 (en) * | 2015-09-16 | 2017-03-16 | Stiftung Caesar Center Of Advanced European Studies And Research | Method for the detection of ligands by means of biosensors |
| CN109627344A (en) * | 2018-12-28 | 2019-04-16 | 深圳先进技术研究院 | CAMP fluorescence probe and its application |
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