WO2021217479A1 - 一种camp荧光探针g-flamp1的应用 - Google Patents

一种camp荧光探针g-flamp1的应用 Download PDF

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WO2021217479A1
WO2021217479A1 PCT/CN2020/087716 CN2020087716W WO2021217479A1 WO 2021217479 A1 WO2021217479 A1 WO 2021217479A1 CN 2020087716 W CN2020087716 W CN 2020087716W WO 2021217479 A1 WO2021217479 A1 WO 2021217479A1
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photon
excitation wavelength
flamp1
camp
probe
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王亮
储军
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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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

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  • the invention belongs to the technical field of biomedical optics and molecular imaging, and specifically relates to the application of a CAMP fluorescent probe G-Flamp1.
  • Cyclic Adenosine Phosphate is the downstream messenger molecule of the G protein-coupled receptor (GPCR) family, the largest drug target at present.
  • GPCR G protein-coupled receptor
  • cAMP fluorescent probes and microscopic imaging of living cells and moving objects are the basic research and drugs of GPCR signaling pathways Important direction of development. Fluorescence imaging of cAMP in living cells refers to expressing cAMP fluorescent probes in the cells, and then using a fluorescent microscope to detect changes in the fluorescent signals (such as intensity) of the probes. Fluorescent probes are the key to cAMP fluorescence imaging analysis. cAMP fluorescent probes are mainly divided into fluorescent resonance energy transfer probes based on fluorescent proteins and probes based on single fluorescent proteins. The latter has a larger dynamic range and is simpler to use.
  • cAMP probes based on a single fluorescent protein are divided into two categories: green and red.
  • the former mainly includes Flamindo2, cADDis and cAMPr, and the latter mainly includes Pink Flamindo, Red cADDis and R-FlincA.
  • the dynamic range of the above probes is relatively small, as shown in the following table.
  • dynamic range (variation amplitude of fluorescence brightness, ⁇ F/F 0 ) is a very important parameter, which is directly related to detection sensitivity. Therefore, improving the dynamic range of the probe in practical applications is of great significance for improving the detection sensitivity.
  • the purpose of the present invention is to provide an application of the cAMP fluorescent probe G-Flamp1.
  • One aspect of the present invention provides an application of a camp fluorescent probe G-Flamp1 in single-photon imaging.
  • the amino acid sequence of G-Flamp1 is shown in SEQ ID NO: 1, and the single-photon excitation wavelength is 430- 470nm.
  • the excitation wavelength of the single photon is 430-450 nm; preferably, the excitation wavelength of the single photon is 450 nm.
  • Another aspect of the present invention provides an application of a camp fluorescent probe G-Flamp1 in two-photon imaging.
  • the amino acid sequence of G-Flamp1 is shown in SEQ ID NO: 1, and the two-photon excitation wavelength is 880 -920nm.
  • the excitation wavelength of the two-photon is 900-920 nm; preferably, the excitation wavelength of the two-photon is 900 nm and 920 nm.
  • Another aspect of the present invention provides an application of the cAMP fluorescent probe G-Flamp1 in the detection of cAMP signals.
  • the single-photon excitation wavelength used in the signal detection is 430-470 nm; the two-photon excitation wavelength is 880-470 nm. 920nm.
  • Another aspect of the present invention provides the application of a cAMP fluorescent probe G-Flamp1 in the detection of cAMP signals in living cells, the single-photon excitation wavelength used in the signal detection is 430-470nm; the two-photon excitation wavelength It is 880-920nm.
  • Another aspect of the present invention provides a method for detecting cAMP fluorescence imaging in living cells, which includes the following steps:
  • the single-photon excitation wavelength used in the imaging analysis is 430-470nm, and the two-photon excitation wavelength is 880-920nm;
  • it comprises the following steps: 1) expressing the cAMP fluorescent probe G-Flamp1 in mammalian cells;
  • Another aspect of the present invention provides an application of the cAMP fluorescent probe G-Flamp1 in the detection of cAMP signals in living brain slices.
  • the single-photon excitation wavelength used in the signal detection is 430-470nm; two-photon excitation The wavelength is 880-920nm.
  • Another aspect of the present invention provides a method for detecting cAMP fluorescence imaging in live brain slices, which includes the following steps:
  • it includes the following steps: 1) Inject a viral vector containing the G-Flamp1 probe gene into the brain region of a mammal;
  • Another aspect of the present invention provides an application of the cAMP fluorescent probe G-Flamp1 in the detection of cAMP signals in moving objects.
  • the single-photon excitation wavelength used in the signal detection is 430-470nm; the two-photon excitation wavelength It is 880-920nm.
  • Another aspect of the present invention provides a method for detecting cAMP fluorescence imaging in a moving object, which includes the following steps:
  • the method includes the following steps: 1) Injecting a virus vector containing the G-Flamp1 probe gene into the target tissue or organ of the mammal;
  • the fluorescence excitation wavelength is usually determined according to the excitation spectrum of this fluorescein, and the peak value is generally taken.
  • the peak value of the excitation spectrum of the G-Flamp1 probe used in the present invention is about 495nm. Therefore, the existing application CN201911251920.X
  • a single photon with an excitation wavelength of ⁇ 480nm is used in the, and the dynamic range of cAMP ( ⁇ F/F 0 ) in cells cultured at a physiological temperature of 37°C is 2.2.
  • This application adopts a single photon with an excitation wavelength of 430-470nm or an excitation wavelength of 880nm-920nm.
  • the dynamic range of cAMP ( ⁇ F/F 0 ) in cells cultured at 37°C physiological temperature is ⁇ 10-15, which has achieved unexpected technical effects and is creative.
  • the beneficial effect of the present invention is that compared with the dynamic range of existing fluorescent probes, the present invention uses 430-470nm single-photon or 880-920nm two-photon to excite the G-Flamp1 probe in cells cultured at a physiological temperature of 37°C.
  • Figure 1 shows the amino acid sequence of #252 and the sequence of G-Flamp1 in Example 1 of the present invention.
  • Figure 2 is the excitation and emission spectra of the purified G-Flamp1 probe in Example 2 of the present invention.
  • the G-Flamp1 probe purified from bacteria is diluted in a HEPES solution of pH 7.3, the final concentration is 2 ⁇ M, and the probe is shown in the figure. Fluorescence excitation spectrum in HEPES solution and saturated concentration of cAMP. The dotted line is the excitation spectrum, and the solid line is the emission spectrum.
  • HEPES buffer (thin line) probe without cAMP in HEPES buffer.
  • Figure 3(A) shows the dynamic range measurement of the purified G-Flamp1 probe in Example 3 of the present invention under single-photon excitation with different excitation wavelengths
  • Figure 3(B) shows the purified G-Flamp1 probe in Example 3 of the present invention
  • the G-Flamp1 probe purified from bacteria was diluted in a HEPES solution of pH 7.3, the final concentration was 2 ⁇ M.
  • the ratio of the fluorescence excitation spectrum with cAMP, F and F 0 are the fluorescence intensity of the probe in the presence of a saturated concentration of cAMP (500 ⁇ M) and without cAMP, respectively.
  • Fig. 4 shows the normalized signal change amplitude measurement of the purified G-Flamp1 probe under different cAMP concentrations in Example 4 of the present invention.
  • the left picture is the test picture under 480nm wavelength excitation, and the right picture is the test under 450nm wavelength excitation. picture.
  • Figure 5 is a comparison diagram of fluorescence brightness of different probes in HEK293T cells in Example 5 of the present invention.
  • Figure 6 shows the response of different probes in HEK293T cells under single-photon excitation in Example 6 of the present invention.
  • Figure 7 shows the response of G-Flamp1 probe in HEK293T cells under two-photon excitation at 920nm in Example 7 of the present invention
  • Lipofectamine was used to transfect HEK293T cells with a plasmid containing G-Flamp1 probe.
  • DMEM cell culture medium of red and serum was starved for 6 hours, and the fluorescence brightness changed after stimulation with 60 ⁇ M Forskolin (Fsk); the two-photon excitation wavelength was 920nm; different curves represent the fluorescence response of different cells; ⁇ F/F 0 is the change in fluorescence intensity The ratio of the initial fluorescence intensity.
  • Figure 8 shows the response of G-Flamp1 probe in HEK293T cells under 900nm two-photon excitation in Example 8 of the present invention
  • Lipofectamine was used to transfect HEK293T cells with a plasmid containing G-Flamp1 probe.
  • the DMEM cell culture medium of red and serum was starved for 6 hours, and the fluorescence intensity changed after stimulation with 60 ⁇ M Forskolin (Fsk); the two-photon excitation wavelength was 900nm; different curves represent the fluorescence response of different cells; ⁇ F/F 0 is the amount of fluorescence intensity change The ratio of the initial fluorescence intensity.
  • Figure 9 is the result of two-photon fluorescence imaging of the neurons in the living brain slice in Example 9 of the present invention.
  • A is the fluorescence brightness chart of the cells before and after the neurons in the brain slice are stimulated by Fsk, the scale is 50 microns;
  • B is ( A) The fluorescence intensity change ( ⁇ F/F 0 ) curve of the representative neuron cell body in A).
  • ⁇ F/F 0 is the ratio of the fluorescence intensity change to the initial fluorescence intensity.
  • the different gray curves are from different cells, and the black curve is the average of the gray curves. .
  • #252 (the amino acid sequence is shown in SEQ ID NO: 2) is mutated to obtain the G-Flamp1 probe (the amino acid sequence is shown in SEQ ID NO: 1), the amino acid sequence of #252 and G-Flamp1 is shown in As shown in Figure 1.
  • the underlined amino acids are the components of the connecting peptide before and after, respectively. Between WG and RV is the circularly rearranged green fluorescent protein sequence (bold part). Before WG is the N-terminal sequence of mICNBD, and after RV is the C-terminal sequence of mICNBD. The bold and slanted amino acids are mutated amino acids.
  • the wavelengths corresponding to the largest peaks of the excitation and emission spectra of the purified G-Flamp1 probe are 495nm and 515nm, respectively; in the HEPES buffer without cAMP, the wavelength of the purified G-Flamp1 probe is The wavelengths corresponding to the largest peaks of the excitation and emission spectra are 502 nm and 517 nm, respectively.
  • the peak size of the fluorescence emission spectrum (the peak value of the probe is 0.106 in the HEPES solution and 1 at the saturated cAMP concentration)
  • the probe fluorescence is brighter than in the HEPES buffer. Increased by 8.4 times.
  • HEPES buffer containing 150mM KCl and 50mM HEPES
  • HisPur Cobalt Resin purchased from Pierce
  • Add 2 ⁇ L HEPES buffer and 2 ⁇ L 30mM cAMP solution final concentration 500 ⁇ M
  • the probe Under single-photon and two-photon excitation, the probe contains saturated cAMP concentration.
  • the ratio of the fluorescence intensity of the needle and the probe without cAMP is shown in Figure 3. It can be seen that the maximum dynamic range is around 450nm and 900nm, respectively.
  • Example 3 The purified probe (2 ⁇ M concentration) in Example 3 was mixed with different concentrations of cAMP to obtain a dose-response curve. As shown in Figure 4, under 480nm and 450nm excitation, its affinity for cAMP is about 2.3 ⁇ M, which meets most of the application requirements.
  • Calcium ion probe GCaMP6s is a classic gene coding probe, which is widely used in live cell and in vivo imaging. Its fluorescence brightness can be used as a reference for this type of gene coding probe.
  • Several gene-encoded probes such as calcium ion probe GCaMP6s, cAMP probe cAMPr/Flamindo2/G-Flamp1, etc. were constructed on the eukaryotic expression vector (CAG promoter), and transfected and cultured by Lipofectamine 2000 kit.
  • HEK293T cells purchased from GE Healthcare Dharmacon) in a glass bottom petri dish.
  • the relative fluorescence intensity of calcium ion probe GCaMP6s and cAMP probe cAMPr/Flamindo2/G-Flamp1 under 480nm excitation are 1, 0.33, 0.28, 0.47, G- Flamp1 is higher than the cAMPr/Flamindo2 probe and is 44% of the widely used GCaMP6s.
  • the relative fluorescence intensity of G-Flamp1 probe under 450nm excitation is 0.22, and its fluorescence brightness is comparable to other green cAMP probes.
  • Probes such as cAMPr, Flamindo2, G-Flamp1, Pink-Flamindo and R-FlincA were constructed on the eukaryotic expression vector (CAG promoter), and the HEK293T cultured in a glass bottom culture dish was transfected by Lipofectamine 2000 kit The cells (purchased from GE Healthcare Dharmacon) were cultured overnight and then starved with serum-free and phenol red-free medium (purchased from GIBCO) for 6 hours. Use the IX83 fluorescence microscope built by our laboratory to detect the brightness of the probe.
  • the excitation wavelength of G-Flamp1 is 440 ⁇ 10nm
  • the fluorescence receiving wavelength is 530 ⁇ 15nm
  • the excitation wavelength of cAMPr and Flamindo2 is 480 ⁇ 15nm
  • the fluorescence receiving wavelength is 530 ⁇ 15nm
  • the excitation wavelength of R-FlincA and Pink-Flamindo is 568 ⁇ 10nm
  • the fluorescence receiving wavelength is 630 ⁇ 25nm.
  • the curve data in Fig. 6 represents: mean value ⁇ standard deviation; ⁇ F/F 0 is the ratio of the change in fluorescence intensity to the initial fluorescence intensity. It can be seen from Figure 6(A) that the signal change range of cAMPr is ⁇ 0.45, the signal change range of Flamindo2 is ⁇ -0.26, the signal change range of Pink-Flamindo is ⁇ 0.89, and the signal change range of R-FlincA is ⁇ 1.28. , It can be seen from Figure 6(B) that the signal variation range of G-Flamp1 is ⁇ 10.
  • G-Flamp1 has the largest signal change amplitude ( ⁇ F/F 0 ), and the dynamic range and sensitivity have been greatly improved.
  • Example 7 The response of G-Flamp1 probe in HEK293T cells under two-photon excitation at 920 nm
  • the G-Flamp1 probe was constructed on a eukaryotic expression vector (CAG promoter), and HEK293T cells (purchased from GE Healthcare Dharmacon) cultured in a glass bottom culture dish were transfected by Lipofectamine 2000 kit. After overnight culture, The cells were starved with serum-free and phenol red-free medium (purchased from GIBCO) for 6 hours. A commercial two-photon microscope was used for imaging analysis. The two-photon excitation wavelength was 920nm. It can be seen that after cells are stimulated by 60 ⁇ M Forskolin (purchased from Biyuntian Biotechnology), the changes in the fluorescence intensity of G-Flamp1 in different cells are shown in Figure 7. .
  • CAG promoter eukaryotic expression vector
  • HEK293T cells purchased from GE Healthcare Dharmacon
  • Example 8 The response of G-Flamp1 probe in HEK293T cells under 900nm two-photon excitation
  • the operation method is the same as that in Example 7, except that the excitation wavelength is changed to 900nm.
  • the experimental results are shown in Figure 8. It can be seen from the figure that the fluorescence intensity of G-Flamp1 in different cells shows different changes, and the average value of ⁇ F/F 0 is 14( The current maximum is 2.2). Among them, different curves represent the fluorescence response of different cells; ⁇ F/F 0 is the ratio of the change in fluorescence intensity to the initial fluorescence intensity.
  • the AAV virus containing the G-Flamp1 gene was injected into the neuron region of the mouse striatum. After 4 weeks, the mice were anesthetized to prepare live brain slices (300 ⁇ m thickness). The brain slices were incubated in artificial cerebrospinal fluid at 33 degrees Celsius for 20-30 minutes, and then incubated at room temperature for 20-30 minutes. Finally, a two-photon fluorescence microscope system was used to monitor the changes in the fluorescence signal of striatal neurons before and after 60 ⁇ M Forskolin stimulation under a 25-fold water mirror and 920nm excitation line. The results are shown in Figure 9.
  • Figure (A) shows the fluorescence brightness of cells before and after Fsk stimulation. Scale: 50 microns.
  • (B) is the fluorescence intensity change ( ⁇ F/F 0 ) curve of the representative neuron cell body in (A).
  • the different gray curves are from different cells, and the black curve is the average value of the gray curve. It can be seen from the figure that ⁇ F/ The average value of F 0 reaches 7, which can be expressed and responded well in neurons.
  • the virus vector containing the probe gene into the mouse brain area of interest; after 3-4 weeks, stimulate the mouse brain cortical neurons to increase or decrease their intracellular cAMP concentration, and use two-photon fluorescence microscopy or intramicroscopic Mirror for imaging analysis.
  • the signal change of the probe can also be detected by a single-photon fluorescence microscope.
  • G-Flamp1 probe in cells cultured at a physiological temperature of 37°C, using 430-470nm single photon or 880-920nm two-photon excitation G-Flamp1 probe has the largest dynamic range ( ⁇ F/F 0 ⁇ 10-15). Increasing can improve the detection sensitivity; in living brain slice neurons, under the stimulation of 60 ⁇ M Forskolin, the average value of ⁇ F/F 0 has also reached 7, which can be expressed and responded well in neurons; in actual use, G -Flamp1 is expressed in mammalian cells or living cells cultured in vitro. Using ordinary fluorescence microscope or two-photon microscope, you can detect whether the cAMP concentration of cells changes after specific stimulation.

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Abstract

提供了一种cAMP荧光探针G-Flamp1在激发波长为430-470nm的单光子或激发波长为880-920nm的双光子成像中的应用，所述G-Flamp1的氨基酸序列如SEQ ID NO：1所示。

Description

一种cAMP荧光探针G-Flamp1的应用 技术领域
本发明属于生物医学光学与分子影像学技术领域，具体涉及一种cAMP荧光探针G-Flamp1的应用。
背景技术
环磷酸腺苷(cAMP)是目前最大药物靶标G蛋白偶联受体(GPCR)家族的下游信使分子，cAMP荧光探针及其活细胞和活动物体显微成像是GPCR信号通路的基础研究和药物开发的重要方向。活细胞中cAMP荧光成像是指将cAMP荧光探针表达在细胞中，然后利用荧光显微镜检测探针荧光信号(如强度)变化。荧光探针是cAMP荧光成像分析的关键。cAMP荧光探针主要分为基于荧光蛋白的荧光共振能量转移探针及基于单个荧光蛋白的探针，后者动态范围较前者大且使用简单。目前基于单个荧光蛋白的cAMP探针分为绿色和红色2小类，前者主要有Flamindo2、cADDis及cAMPr，后者主要有Pink Flamindo、Red cADDis及R-FlincA。在37℃生理温度培养细胞中，上述探针动态范围均较小，如下表所示。实际应用中，动态范围(荧光亮度变化幅度,ΔF/F 0)是很重要的参数，与检测灵敏度直接相关。因此提高探针在实际应用中的动态范围，对于提高探测灵敏度具有重要意义。
Figure PCTCN2020087716-appb-000001
参考文献：
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.
4.CN109627344A，2019.4.16，cAMP荧光探针及其应用
5.CN201911251920.X，荧光亮度宽幅变化的环磷酸腺苷荧光探针
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.
发明内容
为了解决上述背景技术中所提出的技术问题，本发明的目的是提供一种cAMP荧光探针G-Flamp1的应用。
为了达到上述目的，本发明所采用的技术方案为：
本发明一个方面提供了一种cAMP荧光探针G-Flamp1在单光子成像中的应用，所述G-Flamp1的氨基酸序列如SEQ ID NO：1所示，所述单光子的激发波长为430-470nm。
进一步地，所述单光子的激发波长为430-450nm；优选地，所述单光子的激发波长为450nm。
本发明另一个方面提供了一种cAMP荧光探针G-Flamp1在双光子成像中的应用，所述G-Flamp1的氨基酸序列如SEQ ID NO：1所示，所述双光子的激发波长为880-920nm。
进一步地，所述双光子的激发波长为900-920nm；优选地，所述双光子的激发波长为900nm、920nm。
本发明另一个方面提供了一种cAMP荧光探针G-Flamp1在cAMP信号检测中的应用，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
本发明再一个方面提供了一种cAMP荧光探针G-Flamp1在活细胞内cAMP信号检测中的应用，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
本发明再一个方面提供了一种活细胞中cAMP荧光成像检测方法，包括以下步骤：
1)将cAMP荧光探针G-Flamp1表达在哺乳动物细胞中；
2)利用荧光显微镜或者双光子显微镜进行成像分析，检测探针荧光的强度变化，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
优选地，包括以下步骤：1)将cAMP荧光探针G-Flamp1表达在哺乳动物细胞中；
2)刺激哺乳动物细胞升高或者降低其胞内cAMP浓度；
3)利用荧光显微镜或者双光子显微镜进行成像分析，检测上述刺激前后探针荧光的强度变化，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
本发明再一个方面提供了一种cAMP荧光探针G-Flamp1在活体脑片内cAMP信号检测中的应用，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
本发明再一个方面提供了一种活体脑片中cAMP荧光成像检测方法，包括以下步骤：
1)在哺乳动物的脑区注射含G-Flamp1探针基因的病毒载体；
2)待G-Flamp1探针在脑区神经元中表达后，制备活体脑片；
3)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
优选地，包括以下步骤：1)在哺乳动物的脑区注射含G-Flamp1探针基因的病毒载体；
2)待G-Flamp1探针在脑区神经元中表达后，制备活体脑片；
3)刺激哺乳动物脑区中的神经元升高或者降低其胞内cAMP浓度；
4)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析；所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
本发明再一个方面提供了一种cAMP荧光探针G-Flamp1在活动物体内cAMP信号检测中的应用，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
本发明再一个方面提供了一种活动物体中cAMP荧光成像检测方法，包括以下步骤：
1)在哺乳动物的目标组织器官注射含G-Flamp1探针基因的病毒载体；
2)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
优选地，包括以下步骤：1)在哺乳动物的目标组织器官注射含G-Flamp1探针基因的病 毒载体；
2)刺激哺乳动物目标组织器官中的细胞升高或者降低其胞内cAMP浓度；
3)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析；所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
本领域公知荧光激发波长通常根据这种荧光素的激发谱线来确定，一般都取峰值，本发明所采用的G-Flamp1探针的激发光谱的峰值大约在495nm，因此现有申请CN201911251920.X中采用了激发波长～480nm的单光子，37℃生理温度培养细胞中cAMP动态范围(ΔF/F 0)为2.2，本申请通过采用激发波长为430-470nm的单光子或者激发波长为880nm-920nm的双光子，37℃生理温度培养细胞中cAMP动态范围(ΔF/F 0)为～10-15，取得了预料不到的技术效果，具备创造性。
本发明的有益效果是：相较于目前已有荧光探针的动态范围，本发明在37℃生理温度培养细胞中，采用430-470nm单光子或880-920nm双光子激发G-Flamp1探针，获得了目前最大动态范围(ΔF/F 0～10-15，比ΔF/F 0～2.2明显高)，在检测灵敏度方面有了很大提高；在活体脑片神经元中，在60μM Forskolin刺激下，ΔF/F 0平均值也达到了7，可以在神经元中很好的表达和响应；实际使用时，将G-Flamp1表达在离体培养的哺乳动物细胞或活体细胞中，利用普通的荧光显微镜或者双光子显微镜，即可检测细胞受特定刺激后cAMP浓度是否发生改变。
附图说明
图1为本发明实施例1中#252的氨基酸序列及G-Flamp1序列。
图2为本发明实施例2中纯化G-Flamp1探针的激发和发射光谱，从细菌中纯化的G-Flamp1探针稀释在pH 7.3的HEPES溶液中，终浓度为2μM，图示探针在HEPES溶液及饱和浓度cAMP中的荧光激发光谱，虚线为激发光谱，实线为发射光谱，500μM cAMP(粗线)：探针溶液中含cAMP，HEPES缓冲液(细线)：探针在不含cAMP的HEPES缓冲液中。
图3(A)为本发明实施例3中纯化G-Flamp1探针在不同激发波长的单光子激发下的动态范围测定，图3(B)为本发明实施例3中纯化G-Flamp1探针在不同激发波长的双光子激发下的动态范围测定，从细菌中纯化的G-Flamp1探针稀释在pH 7.3的HEPES溶液中，终浓度为2μM，图示探针在饱和浓度cAMP情况下及不含cAMP情况下的荧光激发光谱比值，F，F 0分别为探针在饱和浓度cAMP(500μM)存在时及无cAMP时的荧光强度。
图4为本发明实施例4中纯化G-Flamp1探针在不同cAMP浓度下的归一化的信号变化幅度 测定，左图为480nm波长激发下的测试图，右图为450nm波长激发下的测试图。
图5为本发明实施例5中不同探针在HEK293T细胞中荧光亮度比较图。
图6为本发明实施例6中单光子激发下，不同探针在HEK293T细胞中的响应；(A)利用Lipofectamine分别转染HEK293T细胞含cAMPr、Flamindo2、R-FlincA、Pink-Flamindo探针的质粒，过夜培养后，用不含酚红及血清的DMEM细胞培养液饥饿6小时后，用60μM Forskolin(Fsk)刺激后荧光亮度变化；(B)G-Flamp1探针的响应；G-Flamp1激发波长为440±10nm，荧光接收波长为530±15nm，cAMPr及Flamindo2激发波长为480±15nm，荧光接收波长为530±15nm，R-FlincA及Pink-Flamindo激发波长为568±10nm，荧光接收波长为630±25nm；曲线数据表示：平均值±标准差；ΔF/F 0为荧光强度变化量与初始荧光强度比值。
图7为本发明实施例7中920nm双光子激发下，G-Flamp1探针在HEK293T细胞中的响应；利用Lipofectamine转染HEK293T细胞含G-Flamp1探针的质粒，过夜培养后，用不含酚红及血清的DMEM细胞培养液饥饿6小时后，用60μM Forskolin(Fsk)刺激后荧光亮度变化；双光子激发波长为920nm；不同曲线代表不同细胞的荧光响应；ΔF/F 0为荧光强度变化量与初始荧光强度比值。
图8为本发明实施例8中900nm双光子激发下，G-Flamp1探针在HEK293T细胞中的响应；利用Lipofectamine转染HEK293T细胞含G-Flamp1探针的质粒，过夜培养后，用不含酚红及血清的DMEM细胞培养液饥饿6小时后，用60μM Forskolin(Fsk)刺激后荧光亮度变化；双光子激发波长为900nm；不同曲线代表不同细胞的荧光响应；ΔF/F 0为荧光强度变化量与初始荧光强度比值。
图9为本发明实施例9中活体脑片神经元的双光子荧光成像结果图，(A)为脑片神经元受Fsk刺激前后细胞的荧光亮度图，标尺：50微米；(B)为(A)中代表性神经元胞体的荧光强度变化(ΔF/F 0)曲线，ΔF/F 0为荧光强度变化量与初始荧光强度比值，不同灰色曲线来自不同细胞，黑色曲线为灰色曲线的平均值。
具体实施方式
为了更好地理解本发明的内容，下面结合附图和具体实施方法对本发明内容作进一步说明，但本发明的保护内容不局限于以下实施例。
实施例1 #252的氨基酸序列及G-Flamp1序列
#252(氨基酸序列如SEQ ID NO：2所示)的若干氨基酸进行突变，即得到G-Flamp1探针(氨基酸序列如SEQ ID NO：1所示)，#252及G-Flamp1的氨基酸序列如图1所示。
下划线氨基酸分别为前后连接肽的组分。WG与RV之间是环化重排的绿色荧光蛋白序列(加粗部分)。WG之前为mICNBD的N端序列，RV之后为mICNBD的C端序列。加粗并倾斜的氨基酸为突变的氨基酸。
实施例2 纯化G-Flamp1探针的激发和发射光谱
将G-Flamp1探针表达在细菌中，室温培养2天收集菌体，在pH＝7.3的HEPES缓冲液(含150mM KCl及50mM HEPES)中超声破碎，利用HisPur Cobalt Resin(购自皮尔斯公司)纯化探针，并通过Econo-Pac 10DG脱盐柱(购自美国Bio-Rad公司)将探针溶解在pH＝7.3的HEPES缓冲液中，用BCA试剂盒(购自美国Thermo scientific公司)测定探针浓度。在含120μL 2μM探针溶液的96孔板的2个孔中，分别加入2μL HEPES缓冲液和2μL 30mM cAMP溶液(终浓度为500μM)，然后利用多功能酶标仪Infinite M1000 PRO检测探针激发和发射光谱，如图2所示。在饱和浓度cAMP(500μM)存在情况下，纯化G-Flamp1探针的激发和发射光谱最大峰对应的波长分别是495nm和515nm；在不含cAMP的HEPES缓冲液中，纯化G-Flamp1探针的激发和发射光谱最大峰对应的波长分别是502nm和517nm。通过比较荧光发射谱峰值大小(探针在HEPES溶液中峰值为0.106，在饱和cAMP浓度下为1)，可见在饱和浓度cAMP(500μM)存在情况下，比在HEPES缓冲液中，探针荧光亮度增加了8.4倍。
实施例3 纯化G-Flamp1探针在不同激发波长的单光子和双光子激发下的动态范围测定
将G-Flamp1探针表达在细菌中，室温培养2天收集菌体，在pH＝7.3的HEPES缓冲液(含150mM KCl及50mM HEPES)中超声破碎，利用HisPur Cobalt Resin(购自皮尔斯公司)纯化探针，并通过Econo-Pac 10DG脱盐柱(购自美国Bio-Rad公司)将探针溶解在pH＝7.3的HEPES缓冲液中，用BCA试剂盒(购自美国Thermo scientific公司)测定探针浓度。在含120μL 2μM探针溶液的96孔板的2个孔中，分别加入2μL HEPES缓冲液和2μL 30mM cAMP溶液(终浓度为500μM)，在单光子及双光子激发下，含饱和cAMP浓度的探针与不含cAMP的探针的荧光亮度比值如图3所示，可见最大动态范围分别在450nm及900nm附近。
实施例4 探针对cAMP的亲和力
取实施例3中纯化探针(2μM浓度)分别与不同浓度cAMP混合，得到剂量-反应曲线。如图4所示，在480nm及450nm激发下，其对cAMP亲和力均约为2.3μM，满足大部分的应用需求。
实施例5 不同探针在HEK293T细胞中荧光亮度比较
钙离子探针GCaMP6s为经典的基因编码探针，广泛用于活细胞和活体成像，其荧光亮度可以作为该类基因编码探针的参考。将几种基因编码的探针，如钙离子探针GCaMP6s，cAMP 探针cAMPr/Flamindo2/G-Flamp1等分别构建到真核表达载体上(CAG启动子)，通过Lipofectamine 2000试剂盒转染培养在玻璃底的培养皿中的HEK293T细胞(购买自GE Healthcare Dharmacon公司)。37℃培养48小时后，收集细胞悬液至成像缓冲液中，利用酶标仪检测钙离子探针GCaMP6s，cAMP探针cAMPr/Flamindo2/G-Flamp1在480nm激发下荧光强度以及G-Flamp1探针在450nm激发下荧光强度，检测结果如图5所示，钙离子探针GCaMP6s，cAMP探针cAMPr/Flamindo2/G-Flamp1在480nm激发下相对荧光强度分别是1、0.33、0.28、0.47，G-Flamp1高于cAMPr/Flamindo2探针，为广泛使用的GCaMP6s的44％。G-Flamp1探针在450nm激发下相对荧光强度为0.22，其荧光亮度与其他绿色cAMP探针相当。
实施例6 单光子激发下，不同探针在HEK293T细胞中的响应
将cAMPr、Flamindo2、G-Flamp1、Pink-Flamindo及R-FlincA等探针分别构建到真核表达载体上(CAG启动子)，通过Lipofectamine 2000试剂盒转染培养在玻璃底的培养皿中的HEK293T细胞(购买自GE Healthcare Dharmacon公司)，过夜培养后，用不含血清的、不含酚红的培养基(购自GIBCO公司)饥饿细胞6小时。利用本实验室自行搭建的IX83荧光显微镜检测探针的亮度，G-Flamp1激发波长为440±10nm，荧光接收波长为530±15nm，cAMPr及Flamindo2激发波长为480±15nm，荧光接收波长为530±15nm，R-FlincA及Pink-Flamindo激发波长为568±10nm，荧光接收波长为630±25nm，可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后，各探针的信号变化幅度(ΔF/F 0)，如图6所示。至此完成了哺乳动物细胞内cAMP浓度变化的荧光成像步骤。图6中的曲线数据表示：平均值±标准差；ΔF/F 0为荧光强度变化量与初始荧光强度比值。从图6(A)中可以看出cAMPr的信号变化幅度为～0.45、Flamindo2的信号变化幅度为～-0.26、Pink-Flamindo的信号变化幅度为～0.89、R-FlincA的信号变化幅度为～1.28，从图6(B)中可以看出G-Flamp1的信号变化幅度为～10。经对比可知，可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后，G-Flamp1具最大的信号变化幅度(ΔF/F 0)，在动态范围和灵敏度方面有了很大的提高。
实施例7 920nm双光子激发下，G-Flamp1探针在HEK293T细胞中的响应
将G-Flamp1探针构建到真核表达载体上(CAG启动子)，通过Lipofectamine 2000试剂盒转染培养在玻璃底的培养皿中的HEK293T细胞(购买自GE Healthcare Dharmacon公司)，过夜培养后，用不含血清的、不含酚红的培养基(购自GIBCO公司)饥饿细胞6小时。采用商业化双光子显微镜进行成像分析，双光子激发波长为920nm，可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后，不同细胞中G-Flamp1荧光强度的变化，如图7所示。可见细胞受60μM Forskolin(购自碧云天生物技术公司)刺激后，不同细胞中G-Flamp1荧光 强度展示了不同的变化，其ΔF/F 0平均值为10(目前最高为2.2)。其中，不同曲线代表不同细胞的荧光响应；ΔF/F 0为荧光强度变化量与初始荧光强度比值。
实施例8 900nm双光子激发下，G-Flamp1探针在HEK293T细胞中的响应
操作方法同实施例7，仅改变激发波长为900nm，实验结果如图8所示，由图可知，不同细胞中G-Flamp1荧光强度展示了不同的变化，其ΔF/F 0平均值为14(目前最高为2.2)。其中，不同曲线代表不同细胞的荧光响应；ΔF/F 0为荧光强度变化量与初始荧光强度比值。
实施例9 脑片神经元的双光子荧光成像
将含G-Flamp1基因的AAV病毒注射到小鼠纹状体神经元区域。4周后，将小鼠麻醉，制备活体脑片(300微米厚度)。脑片在33摄氏度的人工脑脊液中孵育20-30分钟，然后在室温孵育20-30分钟。最后利用双光子荧光显微镜系统，在25倍水镜及920nm激发线下，监测纹状体神经元在60μM Forskolin刺激前后的荧光信号改变。结果如图9所示，图(A)中展示了Fsk刺激前后细胞的荧光亮度，标尺：50微米，从图中可以看出Fsk刺激后细胞的荧光亮度显著增强。(B)为(A)中代表性神经元胞体的荧光强度变化(ΔF/F 0)曲线，不同灰色曲线来自不同细胞，黑色曲线为灰色曲线的平均值，从图中可以看出，ΔF/F 0平均值达到了7，可以在神经元中很好的表达和响应。
实施例10 活体检测
在小鼠感兴趣脑区注射含探针基因的病毒载体；3-4周后，刺激小鼠脑区皮质神经元升高或者降低其胞内cAMP浓度，并利用双光子荧光显微镜或者显微内镜进行成像分析。对于组织浅层细胞，也可通过单光子荧光显微镜检测探针的信号变化。
综上，在37℃生理温度培养细胞中，采用430-470nm单光子或880-920nm双光子激发G-Flamp1探针，具最大的动态范围(ΔF/F 0～10-15)，动态范围的增大可以提高检测灵敏度；在活体脑片神经元中，在60μM Forskolin刺激下，ΔF/F 0平均值也达到了7，可以在神经元中很好的表达和响应；实际使用时，将G-Flamp1表达在离体培养的哺乳动物细胞或活体细胞中，利用普通的荧光显微镜或者双光子显微镜，即可检测细胞受特定刺激后cAMP浓度是否发生改变。
以上所述仅为本发明的具体实施方式，不是全部的实施方式，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。

Claims (10)

  1. 一种cAMP荧光探针G-Flamp1在单光子成像中的应用，所述G-Flamp1的氨基酸序列如SEQ ID NO：1所示，其特征在于，所述单光子的激发波长为430-470nm。
  2. 根据权利要求1所述的应用，其特征在于，所述单光子的激发波长为430-450nm；优选地，所述单光子的激发波长为450nm。
  3. 一种cAMP荧光探针G-Flamp1在双光子成像中的应用，所述G-Flamp1的氨基酸序列如SEQ ID NO：1所示，其特征在于，所述双光子的激发波长为880-920nm。
  4. 根据权利要求3所述的应用，其特征在于，所述双光子的激发波长为900-920nm；优选地，所述双光子的激发波长为900nm、920nm。
  5. 一种cAMP荧光探针G-Flamp1在活细胞内cAMP信号检测中的应用，其特征在于，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
  6. 一种活细胞中cAMP荧光成像检测方法，其特征在于，包括以下步骤：
    1)将cAMP荧光探针G-Flamp1表达在哺乳动物细胞中；
    2)利用荧光显微镜或者双光子显微镜进行成像分析，检测探针荧光的强度变化，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
    优选地，包括以下步骤：1)将cAMP荧光探针G-Flamp1表达在哺乳动物细胞中；
    2)刺激哺乳动物细胞升高或者降低其胞内cAMP浓度；
    3)利用荧光显微镜或者双光子显微镜进行成像分析，检测探针荧光的强度变化，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
  7. 一种cAMP荧光探针G-Flamp1在活体脑片内cAMP信号检测中的应用，所述信号检测中所采用的单光子的激发波长为430-470nm；双光子的激发波长为880-920nm。
  8. 一种活体脑片中cAMP荧光成像检测方法，包括以下步骤：
    1)在哺乳动物的脑区注射含G-Flamp1探针基因的病毒载体；
    2)待G-Flamp1探针在脑区神经元中表达后，制备活体脑片；
    3)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
    优选地，包括以下步骤：1)在哺乳动物的脑区注射含G-Flamp1探针基因的病毒载体；
    2)待G-Flamp1探针在脑区神经元中表达后，制备活体脑片；
    3)刺激哺乳动物脑区中的神经元升高或者降低其胞内cAMP浓度；
    4)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析；所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
  9. 一种cAMP荧光探针G-Flamp1在活动物体内cAMP信号检测中的应用，其特征在于，所述信号检测中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
  10. 一种活动物体中cAMP荧光成像检测方法，其特征在于，包括以下步骤：
    1)在哺乳动物的目标组织器官注射含G-Flamp1探针基因的病毒载体；
    2)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm；
    优选地，包括以下步骤：1)在哺乳动物的目标组织器官注射含G-Flamp1探针基因的病毒载体；
    2)刺激哺乳动物目标组织器官中的细胞升高或者降低其胞内cAMP浓度；
    3)利用荧光显微镜、双光子荧光显微镜或者显微内镜进行成像分析，所述成像分析中所采用的单光子的激发波长为430-470nm，双光子的激发波长为880-920nm。
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Citations (2)

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Publication number Priority date Publication date Assignee Title
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
CN109627344A (zh) * 2018-12-28 2019-04-16 深圳先进技术研究院 cAMP荧光探针及其应用

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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
CN109627344A (zh) * 2018-12-28 2019-04-16 深圳先进技术研究院 cAMP荧光探针及其应用

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