CN113567402A - Application of cAMP fluorescent probe G-flash 1 - Google Patents
Application of cAMP fluorescent probe G-flash 1 Download PDFInfo
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Abstract
The invention discloses an application of a cAMP fluorescent probe G-flash 1, in particular to an application of the cAMP fluorescent probe G-flash 1 in single photon imaging with excitation wavelength of 430-470nm or two-photon imaging with excitation wavelength of 880-920nm, wherein the amino acid sequence of the G-flash 1 is shown as SEQ ID NO: 1 is shown. Compared with the dynamic range of the existing fluorescent probe, the invention adopts 430-470nm single photon or 880-920nm two-photon to excite G-flash 1 probe in the cultured cells at the physiological temperature of 37 ℃ to obtain the maximum dynamic range delta F/F010-15, the detection sensitivity is greatly improved; in live brain slice neurons,. DELTA.F/F under 60 μ M Forskolin stimulation0The average value also reaches 7, and the expression and the response in neurons can be good; in practice, G-Flamp1 is expressed in mammalian cells or living cells cultured ex vivo, and the change in cAMP concentration after the cells are specifically stimulated is detected by 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 an application of a cAMP fluorescent probe G-flash 1.
Background
Cyclic adenosine monophosphate (cAMP) is a downstream messenger molecule of the largest drug target G protein-coupled receptor (GPCR) family at present, and cAMP fluorescent probes and microscopic imaging of their living cells and living objects are important directions for fundamental research of GPCR signaling pathways and drug development. cAMP fluorescence imaging in living cells refers to the expression of a cAMP fluorescent probe in a cell, followed by detection of the change in the probe fluorescent signal (e.g., intensity) using a fluorescence microscope.Fluorescent probes are key to the cAMP fluorescence imaging assay. cAMP fluorescent probes are mainly classified into fluorescent protein-based fluorescence resonance energy transfer probes and single fluorescent protein-based probes, the latter having a larger dynamic range than the former and being simple to use. cAMP probes based on a single fluorescent protein are currently classified into the green and Red subclasses 2, the former mainly including Flamido 2, cADDis and cAMPr, and the latter mainly including Pink Flamido, Red cADDis and R-FlincA. The dynamic range of the above probes was small in the cells cultured at physiological temperature of 37 ℃ as shown in the following table. In practical application, dynamic range (amplitude of change in fluorescence luminance,. DELTA.F/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 documents:
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.
CN109627344A, 2019.4.16, cAMP fluorescent probe and application thereof
CN201911251920.X cyclic adenosine monophosphate fluorescent probe with wide-range change of fluorescence brightness
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-flash 1.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of a cAMP fluorescent probe G-flash 1 in single photon imaging, wherein the amino acid sequence of the G-flash 1 is shown as SEQ ID NO: 1, the excitation wavelength of the single photon is 430-470 nm.
Further, the excitation wavelength of the single photon is 430-450 nm; preferably, the excitation wavelength of the single photon is 450 nm.
In another aspect of the invention, the invention provides an application of a cAMP fluorescent probe G-flash 1 in two-photon imaging, wherein the amino acid sequence of the G-flash 1 is shown as SEQ ID NO: 1, the excitation wavelength of the two-photon is 880-920 nm.
Further, the excitation wavelength of the two-photon is 900-920 nm; preferably, the excitation wavelength of the two photons is 900nm and 920 nm.
The invention also provides an application of the cAMP fluorescent probe G-flash 1 in cAMP signal detection, wherein the excitation wavelength of a single photon used in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
The invention further provides an application of the cAMP fluorescent probe G-flash 1 in cAMP signal detection in living cells, wherein the excitation wavelength of a single photon used in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
In another aspect, the present invention provides a method for detecting cAMP fluorescence imaging in living cells, comprising the following steps:
1) expressing cAMP fluorescent probe G-flash 1 in mammalian cells;
2) performing imaging analysis by using a fluorescence microscope or a two-photon microscope, and detecting the fluorescence intensity change of the probe, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
preferably, the method comprises the following steps: 1) expressing cAMP fluorescent probe G-flash 1 in mammalian cells;
2) stimulating mammalian cells to increase or decrease their intracellular cAMP concentration;
3) and (3) carrying out imaging analysis by using a fluorescence microscope or a two-photon microscope to detect the intensity change of the probe fluorescence before and after the stimulation, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm.
The invention further provides an application of the cAMP fluorescent probe G-flash 1 in-vivo brain slice cAMP signal detection, wherein the excitation wavelength of a single photon adopted in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
In another aspect, the invention provides a method for detecting cAMP fluorescence imaging in a brain slice of a living body, which comprises the following steps:
1) injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) preparing a living brain slice after the G-Flamp1 probe is expressed in neurons in a brain area;
3) performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
preferably, the method comprises the following steps: 1) injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) preparing a living brain slice after the G-Flamp1 probe is expressed in neurons in a brain area;
3) stimulating neurons in the brain region of a mammal to raise or lower their intracellular cAMP concentration;
4) performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope; the single photon excitation wavelength used in the imaging analysis is 430-470nm, and the two-photon excitation wavelength is 880-920 nm.
The invention further provides an application of the cAMP fluorescent probe G-flash 1 in cAMP signal detection in a living animal, wherein the excitation wavelength of a single photon adopted in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
In yet another aspect, the present invention provides a method for fluorescence imaging detection of cAMP in a living animal 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 microscope endoscope, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
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 mammalian target tissue organ to increase or decrease their intracellular cAMP concentration;
3) performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope; the single photon excitation wavelength used in the imaging analysis is 430-470nm, and the two-photon excitation wavelength is 880-920 nm.
As is known in the art, the fluorescence excitation wavelength is usually determined according to the excitation line of the fluorescein, and generally takes a peak value, the peak value of the excitation spectrum of the G-Flamp1 probe adopted by the invention is about 495nm, so that the prior application CN201911251920.X adopts a single photon with the excitation wavelength of 480nm, and the cAMP dynamic range (delta F/F) in cells cultured at the physiological temperature of 37 ℃ is adopted0) 2.2, the dynamic range of cAMP (Delta F/F) in cells is cultured at the physiological temperature of 37 ℃ by adopting a single photon with the excitation wavelength of 430-470nm or a two-photon with the excitation wavelength of 880-920nm0) The content is-10-15, the unexpected technical effect is achieved, and the creativity is achieved.
The invention has the beneficial effects that:compared with the dynamic range of the existing fluorescent probe, the invention adopts 430-470nm single photon or 880-920nm two-photon to excite G-flash 1 probe in the cultured cells at the physiological temperature of 37 ℃ to obtain the maximum dynamic range (delta F/F) at present010-15, ratio DeltaF/F02.2 is obviously high), the detection sensitivity is greatly improved; in live brain slice neurons,. DELTA.F/F under 60 μ M Forskolin stimulation0The average value also reaches 7, and the expression and the response in neurons can be good; in practical use, G-Flamp1 is expressed in mammalian cells or living cells cultured in vitro, and a common fluorescence microscope or two-photon microscope is used to detect whether the cAMP concentration changes after the cells are stimulated by a specific stimulus.
Drawings
FIG. 1 shows the amino acid sequence of #252 and the sequence of G-flash 1 in example 1 of the present invention.
FIG. 2 is an excitation and emission spectra of the purified G-flash 1 probe of example 2 of the present invention, wherein the G-flash 1 probe purified from bacteria is diluted in HEPES solution at pH 7.3 to a final concentration of 2 μ M, showing fluorescence excitation spectra of the probe in HEPES solution and cAMP at saturation concentration, with an excitation spectrum in dotted line, an emission spectrum in solid line, and 500 μ M cAMP (bold line): probe solution contained cAMP, HEPES buffer (thin line): the probe was in HEPES buffer without cAMP.
FIG. 3(A) is a graph showing the dynamic range of the purified G-Flamp1 probe in example 3 of the present invention under single photon excitation at different excitation wavelengths, FIG. 3(B) is a graph showing the dynamic range of the purified G-Flamp1 probe in example 3 of the present invention under two-photon excitation at different excitation wavelengths, the G-Flamp1 probe purified from bacteria was diluted in HEPES solution at pH 7.3 to a final concentration of 2. mu.M, and the ratio of fluorescence excitation spectra of the probe in the case of saturated concentration of cAMP and in the case of no cAMP, F0The fluorescence intensity of the probe in the presence of a saturating concentration of cAMP (500. mu.M) and in the absence of cAMP, respectively.
FIG. 4 is a graph of normalized signal amplitude of change measurements of the purified G-Flamp1 probe at different cAMP concentrations in example 4, with the left graph showing the test pattern at 480nm wavelength excitation and the right graph showing the test pattern at 450nm wavelength excitation.
FIG. 5 is a graph showing the comparison of fluorescence intensity of different probes in HEK293T cells in 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) transfecting plasmids containing cAMPr, Flamindo2, R-FlincA and Pink-Flamindo probes of HEK293T cells respectively by using Lipofectamine, after overnight culture, starving for 6 hours by using DMEM cell culture solution without phenol red and serum, and stimulating by using 60 mu M Forskolin (Fsk) to change the fluorescence brightness; (B) response of the G-Flamp1 probe; the excitation wavelength of G-Flamp1 is 440 +/-10 nm, the fluorescence receiving wavelength is 530 +/-15 nm, the excitation wavelengths of cAMPr and Flamido 2 are 480 +/-15 nm, the fluorescence receiving wavelength is 530 +/-15 nm, the excitation wavelengths of R-FlincA and Pink-Flamido are 568 +/-10 nm, and the fluorescence receiving wavelength is 630 +/-25 nm; the curve data represents: mean ± standard deviation; delta F/F0Is the ratio of the fluorescence intensity variation to the initial fluorescence intensity.
FIG. 7 is a graph showing the response of the G-Flamp1 probe in HEK293T cells under 920nm two-photon excitation in example 7 of the present invention; transfecting HEK293T cells with a plasmid containing a G-Flamp1 probe by using Lipofectamine, starving the cells for 6 hours by using DMEM cell culture solution without phenol red and serum after overnight culture, and stimulating the cells by using 60 mu M Forskolin (Fsk) to change the fluorescence brightness; the two-photon excitation wavelength is 920 nm; the different curves represent the fluorescence response of different cells; delta F/F0Is the ratio of the fluorescence intensity variation to the initial fluorescence intensity.
FIG. 8 is a graph showing the response of the G-Flamp1 probe in HEK293T cells under two-photon excitation at 900nm as shown in example 8 of the present invention; transfecting HEK293T cells with a plasmid containing a G-Flamp1 probe by using Lipofectamine, starving the cells for 6 hours by using DMEM cell culture solution without phenol red and serum after overnight culture, and stimulating the cells by using 60 mu M Forskolin (Fsk) to change the fluorescence brightness; the two-photon excitation wavelength is 900 nm; the different curves represent the fluorescence response of different cells; delta F/F0Is the ratio of the fluorescence intensity variation to the initial fluorescence intensity.
Fig. 9 is a two-photon fluorescence imaging result diagram of a live brain slice neuron in example 9 of the present invention, (a) is a fluorescence intensity diagram of cells before and after the brain slice neuron is stimulated by Fsk, and a scale:50 microns; (B) is the change in fluorescence intensity (. DELTA.F/F) of the representative neuronal cell bodies in (A)0) Curve,. DELTA.F/F0The ratio of the change in fluorescence intensity to the initial fluorescence intensity is shown as the difference between the gray curves from different cells and the average of the gray curves.
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings, and the scope of the invention is not limited to the following examples.
EXAMPLE 1#252 amino acid sequence and G-Flamp1 sequence
Amino acid sequence #252 (amino acid sequence shown in SEQ ID NO: 2) is mutated to obtain G-flash 1 probe (amino acid sequence shown in SEQ ID NO: 1), and amino acid sequences #252 and G-flash 1 are shown in FIG. 1.
Underlined amino acids are the components of the connecting peptide, front and back, respectively. Between WG and RV is a circularly rearranged green fluorescent protein sequence (bold). WG is preceded by the N-terminal sequence of mcnbd, RV is followed by the C-terminal sequence of mcnbd. The bold and skewed amino acids are mutated amino acids.
Example 2 excitation and emission spectra of purified G-Flamp1 Probe
The 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 using HisPur Cobalt Resin (available from Pierce), dissolved in HEPES buffer at pH 7.3 using an Econo-Pac 10DG desalting column (available from Bio-Rad, USA), and the probe concentration was determined using a BCA kit (available from Thermo scientific, USA). mu.L of HEPES buffer and 2. mu.L of 30mM cAMP solution (final concentration: 500. mu.M) were added to 2 wells of a 96-well plate containing 120. mu.L of 2. mu.M probe solution, respectively, and then the excitation and emission spectra of the probe were detected using a multi-functional microplate reader Infinite M1000 PRO, as shown in FIG. 2. The wavelengths corresponding to the maximum peaks of the excitation and emission spectra of the purified G-Flamp1 probe in the presence of saturating concentration cAMP (500. mu.M) were 495nm and 515nm, respectively; the wavelengths corresponding to the maximum peaks of the excitation and emission spectra of the purified G-Flamp1 probe in HEPES buffer without cAMP were 502nm and 517nm, respectively. By comparing the peak sizes of the fluorescence emission spectra (peak of 0.106 for probe in HEPES solution and 1 at saturated cAMP concentration), it can be seen that the fluorescence intensity of the probe increases 8.4 times in the presence of saturated cAMP (500. mu.M) compared to that in HEPES buffer.
Example 3 dynamic Range determination of purified G-Flamp1 Probe under Single and two photon excitation at different excitation wavelengths
The 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 using HisPur Cobalt Resin (available from Pierce), dissolved in HEPES buffer at pH 7.3 using an Econo-Pac 10DG desalting column (available from Bio-Rad, USA), and the probe concentration was determined using a BCA kit (available from Thermo scientific, USA). mu.L of HEPES buffer and 2. mu.L of 30mM cAMP solution (final concentration: 500. mu.M) were added to 2 wells of a 96-well plate containing 120. mu.L of 2. mu.M probe solution, respectively, and the ratio of the fluorescence luminance of the probe containing saturated cAMP concentration to that of the probe containing no cAMP under single-photon and two-photon excitation is shown in FIG. 3, and the maximum dynamic ranges are 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 different concentrations of cAMP to obtain a dose-response curve. As shown in FIG. 4, under excitation at 480nm and 450nm, the affinity to cAMP is about 2.3. mu.M, which satisfies most application requirements.
Example 5 comparison of fluorescence intensity of different probes in HEK293T cells
The calcium ion probe GCaMP6s is a classical gene coding probe, is widely used for live cell 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. Probes encoded by several genes, such as calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamido 2/G-Flamp1, etc., were constructed separately on eukaryotic expression vectors (CAG promoter), and HEK293T cells (purchased from GE Healthcare Dharmacon) cultured in a glass-bottomed dish were transfected by Lipofectamine 2000 kit. After culturing at 37 ℃ for 48 hours, the cell suspension was collected into an imaging buffer, and the fluorescence intensity of calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamiddo 2/G-Flamp1 under 480nm excitation and G-Flamp1 probe under 450nm excitation were detected by a microplate reader, as shown in FIG. 5, the fluorescence intensity of calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamiddo 2/G-Flamp1 under 480nm excitation was 1, 0.33, 0.28, 0.47, and G-Flamp1 was higher than that of cAMPr/Flamiddo 2 probe and was 44% of that of GCaMP6s, which is widely used. The relative fluorescence intensity of the G-Flamp1 probe under 450nm excitation was 0.22, which is comparable to that of other green cAMP probes.
Example 6 response of different probes in HEK293T cells under Single photon excitation
Probes such as cAMPr, Flamido 2, G-Flamp1, Pink-Flamido, and R-FlincA were constructed on eukaryotic expression vectors (CAG promoters), and HEK293T cells (purchased from GE Healthcare Dharmacon) cultured in a glass-bottomed dish were transfected with a Lipofectamine 2000 kit, and after overnight culture, the cells were starved for 6 hours with a serum-free and phenol red-free medium (purchased from GIBCO). The luminance of the probe is detected by using an IX83 fluorescence microscope self-built in the laboratory, the excitation wavelength of G-flash 1 is 440 +/-10 nm, the fluorescence receiving wavelength is 530 +/-15 nm, the excitation wavelengths of cAMPr and Fluminono 2 are 480 +/-15 nm, the fluorescence receiving wavelength is 530 +/-15 nm, the excitation wavelengths of R-FlincA and Pink-Fluminoo are 568 +/-10 nm, the fluorescence receiving wavelength is 630 +/-25 nm, and after visible cells are stimulated by 60 mu M Forskolin (purchased from Biyunnan Biotech company), the signal change amplitude (delta F/F) of each probe is0) As shown in fig. 6. This completes the fluorescence imaging step of changes in cAMP concentration in mammalian cells. The curve data in fig. 6 represents: mean ± standard deviation; delta F/F0Is the ratio of the fluorescence intensity variation to the initial fluorescence intensity. FIG. 6(A) shows that the signal change width of cAMPr is-0.45, the signal change width of Flamido 2 is-0.26, the signal change width of Pink-Flamido is-0.89, the signal change width of R-FlancA is-1.28, and FIG. 6(B) shows that the signal change width of G-Flamp1 is-10. By comparison, it can be seen that the cells received 60 μ M Forskolin (purchased from Biyun Tiansheng)Technology Co.) stimulation, G-flash 1 showed the greatest amplitude of signal change (Δ F/F)0) The dynamic range and sensitivity are greatly improved.
Example response of G-flash 1 Probe in HEK293T cells under 7920 nm two-photon excitation
The G-Flamp1 probe was constructed onto a eukaryotic expression vector (CAG promoter), HEK293T cells (purchased from GE Healthcare Dharmacon) cultured in a glass-bottomed dish were transfected by a Lipofectamine 2000 kit, and after overnight culture, the cells were starved for 6 hours with serum-free, phenol red-free medium (purchased from GIBCO). 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 different cells was observed after the cells were stimulated with 60 μ M Forskolin (available from bi yunnan biotechnology), as shown in fig. 7. It can be seen that the G-flash 1 fluorescence intensity in different cells shows different changes after the cells are stimulated by 60 μ M Forskolin (purchased from Biyuntian Biotech Co., Ltd.), and the delta F/F of the changes0The average value is 10 (currently up to 2.2). Wherein the different curves represent the fluorescence response of different cells; delta F/F0Is the ratio of the fluorescence intensity variation to the initial fluorescence intensity.
Example response of G-Flamp1 Probe in HEK293T cells under 8900 nm two-photon excitation
The procedure was the same as in example 7 except that the excitation wavelength was changed to 900nm, and the experimental results are shown in FIG. 8, which shows that the fluorescence intensity of G-Flamp1 in different cells shows different changes, and Δ F/F thereof0The average value was 14 (currently up to 2.2). Wherein the different curves represent the fluorescence response of different cells; delta F/F0Is the ratio of the fluorescence intensity variation 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 striatal neuronal region of mice. After 4 weeks, the mice were anesthetized and live brain slices (300 μm thickness) were prepared. Brain slices were incubated in artificial cerebrospinal fluid at 33 ℃ for 20-30 minutes, then at room temperature for 20-30 minutes. Finally, by utilizing a two-photon fluorescence microscope system,changes in the fluorescence signal of striatal neurons before and after 60 μ M Forskolin stimulation were monitored under 25-fold water scope and 920nm excitation line. The results are shown in fig. 9, where the fluorescence intensity of the cells before and after Fsk stimulation is shown in panel (a), scale: 50 microns, it can be seen that the fluorescence intensity of the cells after Fsk stimulation is significantly enhanced. (B) Is the change in fluorescence intensity (. DELTA.F/F) of the representative neuronal cell bodies in (A)0) Curves, different grey curves from different cells, and black curves as the mean of the grey curves, it can be seen from the figure that Δ F/F0The average value reaches 7, and the expression and the response in neurons can be good.
Example 10 in vivo assay
Injecting a virus vector containing a probe gene into a mouse brain region of interest; after 3-4 weeks, cortical neurons in the mouse brain region are stimulated to increase or decrease their intracellular cAMP concentration, and analyzed by imaging using a two-photon fluorescence microscope or endomicroscopy. For tissue superficial cells, the signal change of the probe can also be detected by a single photon fluorescence microscope.
In conclusion, in the cell culture at the physiological temperature of 37 ℃, the G-flash 1 probe is excited by adopting 430-470nm single photon or 880-920nm two-photon, and the maximum dynamic range (delta F/F) is obtained010-15), the increase of the dynamic range can improve the detection sensitivity; in live brain slice neurons,. DELTA.F/F under 60 μ M Forskolin stimulation0The average value also reaches 7, and the expression and the response in neurons can be good; in practical use, G-Flamp1 is expressed in mammalian cells or living cells cultured in vitro, and a common fluorescence microscope or two-photon microscope is used to detect whether the cAMP concentration changes after the cells are stimulated by a specific stimulus.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> application of cAMP fluorescent probe G-flash 1
<130> CP120010177C
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 422
<212> PRT
<213> Artificial sequence
<400> 1
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Trp Gln Leu Val Ala Ala Val Pro Leu Phe Gln Lys Leu Gly Pro Ala
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Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile
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Arg His Asn Val Glu Gly Gly Gly Val Gln Leu Ala Tyr His Tyr Gln
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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 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 (10)
1. The application of a cAMP fluorescent probe G-flash 1 in single photon imaging is disclosed, wherein the amino acid sequence of the G-flash 1 is shown as SEQ ID NO: 1, wherein the excitation wavelength of the single photon is 430-470 nm.
2. The use according to claim 1, wherein the excitation wavelength of said single photon is 430-450 nm; preferably, the excitation wavelength of the single photon is 450 nm.
3. The application of a cAMP fluorescent probe G-flash 1 in two-photon imaging, wherein the amino acid sequence of the G-flash 1 is shown as SEQ ID NO: 1, wherein the two-photon excitation wavelength is 880-920 nm.
4. The use of claim 3, wherein the two-photon excitation wavelength is 900-920 nm; preferably, the excitation wavelength of the two photons is 900nm and 920 nm.
5. An application of a cAMP fluorescent probe G-flash 1 in cAMP signal detection in living cells is characterized in that the excitation wavelength of a single photon adopted in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
6. A method for detecting cAMP fluorescence imaging in living cells, which is characterized by comprising the following steps:
1) expressing cAMP fluorescent probe G-flash 1 in mammalian cells;
2) performing imaging analysis by using a fluorescence microscope or a two-photon microscope, and detecting the fluorescence intensity change of the probe, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
preferably, the method comprises the following steps: 1) expressing cAMP fluorescent probe G-flash 1 in mammalian cells;
2) stimulating mammalian cells to increase or decrease their intracellular cAMP concentration;
3) and (3) carrying out imaging analysis by using a fluorescence microscope or a two-photon microscope to detect the intensity change of the fluorescence of the probe, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm.
7. An application of a cAMP fluorescent probe G-flash 1 in-vivo brain slice cAMP signal detection, wherein the excitation wavelength of a single photon adopted in the signal detection is 430-470 nm; the excitation wavelength of the two-photon is 880-920 nm.
8. A cAMP fluorescence imaging detection method in a live brain slice comprises the following steps:
1) injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) preparing a living brain slice after the G-Flamp1 probe is expressed in neurons in a brain area;
3) performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
preferably, the method comprises the following steps: 1) injecting a virus vector containing a G-Flamp1 probe gene into a brain region of a mammal;
2) preparing a living brain slice after the G-Flamp1 probe is expressed in neurons in a brain area;
3) stimulating neurons in the brain region of a mammal to raise or lower their intracellular cAMP concentration;
4) performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope; the single photon excitation wavelength used in the imaging analysis is 430-470nm, and the two-photon excitation wavelength is 880-920 nm.
9. An application of a cAMP fluorescent probe G-flash 1 in cAMP signal detection in a living animal is characterized in that the excitation wavelength of a single photon used in the signal detection is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm.
10. A method for fluorescence imaging detection of cAMP in a living animal 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 microscope endoscope, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm;
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 mammalian target tissue organ to increase or decrease their intracellular cAMP concentration;
3) and performing imaging analysis by using a fluorescence microscope, a two-photon fluorescence microscope or a microscope endoscope, wherein the excitation wavelength of a single photon adopted in the imaging analysis is 430-470nm, and the excitation wavelength of a two-photon is 880-920 nm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2015066706A2 (en) * | 2013-11-04 | 2015-05-07 | 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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| WO2015066706A2 (en) * | 2013-11-04 | 2015-05-07 | 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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