CAMP fluorescent probe G-Flamp3 and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly provides a cAMP fluorescent probe G-Flamp3, and the amino acid sequence of the G-Flamp3 is as shown in SEQ ID NO: 2. The cAMP fluorescent probe G-Flamp3 provided by the invention is a green cAMP probe with higher sensitivity, and when the cAMP fluorescent probe G-Flamp3 is used for culturing mammalian cells at 37 DEG C, the fluorescent brightness of the cAMP fluorescent probe G-Flamp3 is further improved compared with that of existing green fluorescent probes G-Flamp1 and G-Flamp2. In cells cultured at 37 DEG C, the dynamic range (delta F / F0) of the fluorescence change of the G-Flamp3 is larger than that of the G-Flamp1 and the G-Flamp2 under the condition that the cAMP is slightly increased and decreased. In actual use, the cAMP is expressed in mammalian cells, and the change of the concentration of the cAMP after the cells are specifically stimulated can be detected by using a common fluorescence microscope.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a cAMP fluorescent probe G-Flamp3 and its application. Background Art

[0002] Cyclic adenosine monophosphate (cAMP) is a downstream messenger molecule of the largest drug target G protein-coupled receptor (GPCR) family currently. cAMP fluorescence imaging at the cellular and in vivo levels is an important direction for basic research on GPCR signaling pathways and drug development. cAMP fluorescent probes are mainly divided into fluorescence 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 than the former. Currently, cAMP probes based on single fluorescent proteins are divided into two categories: green and red. The former mainly includes Flamindo2, cADDis, cAMPr, G-Flamp1, G-Flamp2, and G-Flamp2b, and the latter mainly includes R-Flamp1m, R-Flamp2m, Pink Flamindo, Red cADDis, and R-FlincA. According to the experimental results of existing research, it is known that the affinity of some intracellular cAMP effectors for cAMP is about several hundred nanomoles. There is spontaneous oscillation of cAMP in some cells, and the fluctuation of its cAMP concentration is relatively low. Currently, the high-performance G-Flampl and G-Flamp2 probes based on circularly permuted green fluorescent proteins have an affinity for cAMP of 1-2 μM. Therefore, it is necessary to develop probes with higher affinity to meet the needs of sensitive detection of cAMP in these cells. In summary, developing high-performance cAMP fluorescent probes of different colors (such as green and red), and improving their affinity, brightness, and dynamic range in practical applications are of great significance for improving detection sensitivity and multicolor imaging. Summary of the Invention

[0003] The object of the present invention is to develop probes with higher affinity to meet the needs of sensitive detection of cAMP in cells.

[0004] To this end, the present invention provides a cAMP fluorescent probe G-Flamp3, and the amino acid sequence of the G-Flamp3 is shown in SEQ ID NO: 2.

[0005] SEQ ID NO: 2

[0006] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRREDFVRNWQLVAAVPLFQKLDPAVLDEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYITADKQNNGIKANFEIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEYVTAAGITLGMDELYKGGTGGSMVRKEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLSLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNNSNPVKLGPGAFFGEMALISGEPRVATVIAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGP

[0007] Specifically, the above-mentioned fluorescent probe G-Flamp3 is used under the excitation wavelength of 450 nm for single photons.

[0008] The cAMP fluorescent probe G-Flamp3 provided by the present invention can be used for detecting cAMP, detecting the changes of cAMP in living cells and / or in vivo of live animals, and screening GPCR-targeted drugs.

[0009] The present invention provides a method for detecting cAMP fluorescence imaging in living cells, comprising the following steps:

[0010] (1) Culturing living cells, when the cell density reaches 60%, transfecting with the G-Flamp3 plasmid;

[0011] (2) Culturing the transfected cells overnight, after starving the cells for 4 h, changing the culture medium to a colorless and transparent buffer solution;

[0012] (3) Under the excitation wavelength of 450 nm for single photons, performing fluorescence microscopy imaging analysis.

[0013] The present invention also provides a method for detecting cAMP fluorescence imaging in mammalian cells for non-diagnostic purposes, comprising the following steps:

[0014] (1) Culturing mammalian cells, the culture medium is DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature is 37 °C, and the CO2 content is 5%; when the cell density reaches 60%, transfecting with the G-Flamp3 plasmid;

[0015] (2) The transfected mammalian cells are cultured overnight. After starving the cells for 4 h with a medium without serum and phenol red, the medium is replaced with a colorless and transparent buffer for fluorescence imaging.

[0016] (3) Fluorescence microscopy imaging analysis is performed, where the excitation wavelength of single photons is 450 nm.

[0017] The present invention also provides a kit containing the cAMP fluorescent probe G-Flamp3 described above.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The cAMP fluorescent probe G-Flamp3 provided by the present invention is a green cAMP probe with higher sensitivity. In mammalian cells cultured at 37 °C, the fluorescence brightness is further improved compared with the existing green fluorescent probes G-Flamp1 and G-Flamp2. In cells cultured at 37 °C, the dynamic range (ΔF / F 0 ) of the fluorescence change of G-Flamp3 under weak elevation and reduction of cAMP is larger than that of G-Flamp1 and G-Flamp2. In actual use, when it is expressed in mammalian cells, the change in cAMP concentration after the cells are stimulated by a specific stimulus can be detected using an ordinary fluorescence microscope.

[0020] The present invention will be further described in detail below with reference to the accompanying drawings. Description of the Drawings

[0021] Figure 1 It is a comparison of the affinity curves of the G-Flamp1, G-Flamp2, and G-Flamp3 probes in the examples of the present invention; among them, the solid line is G-Flamp1, the dotted line with circles is G-Flamp2, and the dotted line with squares is G-Flamp3.

[0022] Figure 2 It is the fluorescence excitation and emission spectra of the G-Flamp3 probe in the examples of the present invention; among them, the dotted line represents the excitation spectrum, the solid line represents the emission spectrum; +cAMP indicates that the probe binds to cAMP, and -cAMP indicates no cAMP.

[0023] Figure 3It is the comparison of the brightness of G-Flamp1, G-Flamp2, and G-Flamp3 in HEK293T cells under single-photon excitation in the embodiments of the present invention; the left figure is the comparison of the resting fluorescence intensities of G-Flamp1, G-Flamp2, and G-Flamp3 at an excitation wavelength of 450 nm, and the right figure is the comparison of the resting fluorescence intensities of G-Flamp1, G-Flamp2, and G-Flamp3 at an excitation wavelength of 480 nm.

[0024] Figure 4 It is the response of G-Flamp1, G-Flamp2, and G-Flamp3 probes in HEK293T cells in the embodiments of the present invention; among them, A is the fluorescence change of G-Flamp1, G-Flamp2, and G-Flamp3 probes in HEK293T cells under the stimulation of 15 nM Iso; B is the response of G-Flamp2 and G-Flamp3 probes in HEK293T cells under the stimulation of 1 μM NA; C is the fluorescence change of G-Flamp1, G-Flamp2, and G-Flamp3 probes in HEK293T cells under the stimulation of 10 μM Qui; the excitation wavelength is 450 nm, and different curves represent the responses of different probes (mean ± standard error). Detailed implementation manners

[0025] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, those of ordinary skill in the technical field to which the present invention belongs will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0026] The effect of the cAMP fluorescent probe G-Flamp3 of the present invention is studied below through specific examples.

[0027] Example 1: Construction of G-Flamp3

[0028] By mutating some amino acids of G-Flamp2, the G-Flamp3 probe is obtained. The amino acid sequence of G-Flamp2 is shown in SEQ ID NO: 1.

[0029] SEQ ID NO: 1

[0030] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYITADKQNNGIKANFEIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLSLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNRVNPVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA

[0031] The amino acid sequence of G-Flamp3 is shown in SEQ ID NO: 2.

[0032] SEQ ID NO: 2

[0033] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRREDFVRNWQLVAAVPLFQKLDPAVLDEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYITADKQNNGIKANFEIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEYVTAAGITLGMDELYKGGTGGSMVRKEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLSLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNNSNPVKLGPGAFFGEMALISGEPRVATVIAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGP

[0034] The second amino acid R of SEQ ID NO: 1 and SEQ ID NO: 2 can be removed.

[0035] Example 2: Excitation and emission spectra of the G-Flamp3 probe

[0036] Express the G-Flamp1, G-Flamp2, and G-Flamp3 probes in bacteria respectively, culture the bacteria at room temperature for 3 days, collect the bacterial cells, ultrasonically disrupt them in HEPES buffer (containing 150 mM KCl and 50 mM HEPES) at pH = 7.2, and then centrifuge to obtain the supernatant (containing the probe).

[0037] Take 120 μL of the probe solution and use a multi-functional microplate reader Infinite M1000 PRO to detect the response of the probe to different cAMP concentrations. The excitation wavelength of fluorescence is 450 nm, and the receiving wavelength is 515 nm. Obtain the ΔF / F 0 Curve with the cAMP concentration, and then the dynamic ranges of G-Flamp1, G-Flamp2, and G-Flamp3 at different cAMP concentrations can be compared, and the dissociation constant Kd can be obtained. The results are as Figure 1 shown. At the same time, the fluorescence excitation and emission spectra can be obtained as Figure 3 shown.

[0038] It can be seen from the figure that the dynamic range ΔF / F of the fluorescence change of G-Flamp3 provided by the present invention under weak elevation and reduction of cAMP 0 is larger and the detection sensitivity is higher.

[0039] The amino acid sequence of G-Flamp1 is shown in SEQ ID NO: 3.

[0040] SEQ ID NO: 3

[0041] MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPMGFYQEVRRGDFVRNWQLVAAVPLFQKLGPAVLVEIVRALRARTVPAGAVICRIGEPGDRMFFVVEGSVSVATNWGNVYITADKQKNGIKANFKIRHNVEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYIQERTIVFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNRVNPVELGPGAFFGEMALISGEPRVATVSAATTVSLLSLHSADFQMLCSSSPEIAEIFRKTALERRGAAASA

[0042] The second amino acid R in SEQ ID NO: 3 can be removed.

[0043] Example 3: Brightness comparison of G-Flamp1, G-Flamp2 and G-Flamp3 in HEK293T cells

[0044] Culture HEK293T cells in a 6-well plate with DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at a culture temperature of 37 °C and a CO 2 content of 5%.

[0045] Transfect plasmids expressing G-Flamp1, G-Flamp2 and G-Flamp3 of the same mass into cells in different wells using the Lipofectamine 2000 kit. After 40 hours, wash the cells once with colorless and transparent live cell imaging buffer, then resuspend the cells in 200 μL of live cell imaging buffer, transfer them to a 96-well microplate reader, and after standing at 37 °C for 10 minutes, detect the fluorescence intensity of the cells. The results are as Figure 3 shown.

[0046] The fluorescence brightness of the G-Flamp3 probe is further increased compared to G-Flamp1 and G-Flamp2 in cells cultured at 37 °C.

[0047] Example 4: Responses of G-Flamp1, G-Flamp2, and G-Flamp3 probes in HEK293T cells under single-photon excitation

[0048] HEK293T cells were cultured in glass-bottomed dishes with DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin at a culture temperature of 37 °C and a CO 2 content of 5%. When the cell density was about 60%, G-Flamp1, G-Flamp2, and G-Flamp3 plasmids were transfected using the Lipofectamine 2000 kit. After overnight culture, the cells were starved for 4 hours with a serum- and phenol red-free medium (purchased from GIBCO), and then the medium was replaced with a colorless and transparent live cell imaging buffer. The cells were collected and adhered to the microplate, and the responses of the probes were detected using the multimode microplate reader Infinite M1000 PRO.

[0049] After drug stimulation, the changes in the fluorescence intensities of G-Flamp1, G-Flamp2, and G-Flamp3 in the cells are as Figure 4 shown. Using 15 nM isoproterenol (Iso) produced a small amount of cAMP in HEK293T cells, as shown in Figure 4 A. Using 1 μM NA produced a small amount of cAMP in HEK293T cells, as shown in Figure 4 B. Activating intracellular D2R with 10 μM Qui inhibited adenylate cyclase, thereby reducing the intracellular cAMP concentration, as shown in Figure 4 C. Thus, the fluorescence imaging step of the change in intracellular cAMP concentration in mammalian cells was completed.

[0050] As can be seen from Figure 4 this, the G-Flamp3 probe can obtain the largest dynamic range among current green cAMP probes using the optimal excitation wavelength in cells cultured at 37 °C, and there has been a great improvement in sensitivity.

[0051] In summary, the cAMP fluorescent probe G-Flamp3 provided by the present invention is a green cAMP probe with higher sensitivity. In mammalian cells cultured at 37 °C, the fluorescence brightness is further improved compared to the existing green fluorescent probes G-Flamp1 and G-Flamp2. In cells cultured at 37 °C, the dynamic range (ΔF / F 0)It is larger than G-Flamp1 and G-Flamp2. In actual use, when it is expressed in mammalian cells, the change in cAMP concentration after the cells are stimulated by a specific stimulus can be detected using an ordinary fluorescence microscope.

[0052] The above examples are only illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. Any design identical or similar to the present invention falls within the protection scope of the present invention.

Claims

1. A cAMP fluorescent probe G-Flamp3, characterized in that: the amino acid sequence of the G-Flamp3 is shown as SEQ ID NO:

2.

2. The cAMP fluorescent probe G-Flamp3 according to claim 1, characterized in that: the fluorescent probe G-Flamp3 is used under a single-photon excitation wavelength of 450 nm.

3. The application of the cAMP fluorescent probe G-Flamp3 according to any one of claims 1 or 2 in detecting cAMP.

4. The application of the cAMP fluorescent probe G-Flamp3 according to any one of claims 1 or 2 in detecting changes in cAMP in living cells and / or in vivo in animals.

5. A method for detecting cAMP fluorescence imaging in living cells, characterized in that it comprises the following steps: (1) Culturing living cells, when the cell density reaches 60%, transfecting with the G-Flamp3 plasmid; (2) Culturing the transfected cells overnight, starving the cells for 4 h, and then changing the culture medium to a colorless and transparent buffer solution; (3) Performing fluorescence microscopy imaging analysis under a single-photon excitation wavelength of 450 nm.

6. A method for detecting cAMP fluorescence imaging in mammalian cells for non-diagnostic purposes, characterized in that it comprises the following steps: (1) Mammalian cell culture, the culture medium is DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin, the culture temperature is 37 °C, and the CO 2 content is 5%; when the cell density reaches 60%, transfect with the G-Flamp3 plasmid; (2) Culturing the transfected mammalian cells overnight, starving the cells for 4 h with a culture medium without serum and phenol red, and then changing the culture medium to a colorless and transparent buffer solution for fluorescence imaging; (3) Performing fluorescence microscopy imaging analysis, wherein the single-photon excitation wavelength is 450 nm.

7. The application of the cAMP fluorescent probe G-Flamp3 according to any one of claims 1 or 2 in GPCR-targeted drug screening.

8. A kit comprising the cAMP fluorescent probe G-Flamp3 according to any one of claims 1 or 2.

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