Biosensor for detecting csk kinase activity in cells and detection method thereof

Abstract

Provided are a biosensor for detecting Csk kinase activity in cells and a detection method thereof, relating to the technical field of biosensors. The biosensor fuses N-terminal ECFP, an SH2 domain of Src, a flexible linker having 15 amino acids, a substrate polypeptide, and C-terminal YPet; the substrate polypeptide is selected from one of a Src kinase C-terminal tail peptide chain FTSTEPQYQPGENL, a Fyn kinase C-terminal tail peptide chain FTATEPQYQPGENL, and a Csk specific substrate peptide EEEIYFFF. An FRET biosensor provided by the present invention and relating to a fluorescence resonance energy transfer (FRET) technology and gene encoding provides a powerful tool for visualizing signal molecules in living cells with high temporal-spatial resolution, can specifically visualize the dynamic state of the Csk kinase in the cells, and provides a visualization tool for further exploring the action mechanism of in-vivo Csk.

Description

A biosensor for detecting intracellular Csk kinase activity and its detection method Technical Field

[0001] The present invention belongs to the technical field of biosensors, in particular to a biosensor for detecting intracellular Csk kinase activity and a detection method thereof. Background Art

[0002] CSK participates in a variety of cellular functions, such as apoptosis, proliferation, cytoskeletal reorganization, cell migration, invasion, and angiogenesis, primarily through the regulation of SFKs. It is also involved in preventing cancer progression, as SFKs are associated with cell differentiation, cancer staging, and the development of metastasis. Despite the importance of CSK function in regulating normal cell growth, imaging tools that can directly visualize CSK activity are lacking.

[0003] Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a biosensor for detecting intracellular Csk kinase activity and its detection method. Based on the FRET principle and utilizing molecular cloning technology, a genetically encoded biosensor was developed to detect the activity of C-terminal Src kinase (Csk) in cells. The present invention designed biosensors for three different substrates, selected the optimal substrate, and then added a membrane-localizing tag to the C-terminus or N-terminus to screen for biosensors with high sensitivity and good specificity. Fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide powerful tools for visualizing signaling molecules in living cells with high spatiotemporal resolution.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A biosensor for detecting intracellular Csk kinase activity and a detection method thereof. The biosensor sequentially fuses ECFP at the N-terminus, the SH2 domain of Src, a flexible linker with 15 amino acids, a substrate polypeptide, and YPet at the C-terminus; the substrate polypeptide is selected from one of the following: Src kinase C-terminal tail peptide chain FTSTEPQYQPGENL, Fyn kinase C-terminal tail peptide chain FTATEPQYQPGENL, and Csk-specific substrate peptide EEEIYFFF.

[0007] A detection method of the above-mentioned biosensor for detecting intracellular Csk kinase activity specifically comprises the following steps:

[0008] Step S1, molecular cloning construction, includes:

[0009] Step S11, constructing a PCR reaction system;

[0010] Step S12, enzymatic digestion of the target fragment;

[0011] Step S13, the target fragment after enzyme digestion and the vector are connected using T4 DNA ligase;

[0012] Step S14, competent state transformation;

[0013] Step S15, identification of the ligated plasmid;

[0014] Step S2, transfection of mammalian cells with FRET probe: transfection of the Csk FRET probe into cells using liposome transfection;

[0015] Step S3, quantitative and statistical analysis of FRET image data: Data analysis was performed using the FRET image analysis software Fluocell 6.0.0, developed using the MATLAB software platform. Experimental analysis data were expressed as "mean + standard deviation" and analyzed using the statistical analysis software Origin 8.0 and GraphPad Prism 6.0. The differences between the data groups were compared using the t-test, and P < 0.05 indicated that the difference was statistically significant.

[0016] Furthermore, the competent transformation step S14 specifically includes the following steps:

[0017] Step S141: Take out a 50 μL tube of DH5α competent E. coli from a -80°C refrigerator and quickly freeze and thaw on ice;

[0018] Step S142: Add 0.5-2 μL of plasmid DNA to the competent E. coli and place on ice for 30 minutes;

[0019] Step S143, heat shock: place in a 42°C metal bath for 30-90 seconds;

[0020] Step S144, placing the EP tube on ice for 2 minutes;

[0021] Step S145: adding 1 mL of resistance-free LB medium;

[0022] Step S146: incubate at 37°C with shaking at 250 rpm for 1 h;

[0023] Step S147: Take out 250 μL of the bacterial solution and apply it to the LB agar plate with the corresponding resistance of the plasmid DNA. If you want to increase the density of the bacteria on the plate, centrifuge the bacteria solution at 4000-8000 rpm for 1 minute, resuspend it in 250 μL of LB medium, and apply the entire bacterial solution to the plate;

[0024] Step S148: Place the culture plate upside down in a 37°C constant temperature incubator overnight;

[0025] Step S149: After the colonies grow, remove the agar plate; pick a single clone on a clean bench and place it in LB medium containing the corresponding resistance for culture and amplification; after 12 to 16 hours, extract the plasmid according to the corresponding steps.

[0026] Furthermore, the step S2 of transfecting mammalian cells with FRET probes comprises culturing the cells in a 24-well cell culture plate, and transfecting or co-transfecting Csk FRET probes, CSK plasmids, Fyn plasmids, Src plasmids, and PTPα plasmids, specifically comprising the following steps:

[0027] Step S21, culturing ASMC / MEF / Hela in 10% FBS DMEM low-glucose medium, with a small number of passages. When the cell density is 80-90%, the cells are seeded in a 24-well plate in advance. Preferably, the cell density is about 60% during transfection;

[0028] Step S22: According to the Lipofectamine 3000 liposome transfection method, each well was co-transfected with 1 μg of CskFRET plasmid DNA, 0.3 μg of CSK plasmid, or 0.3 μg of Fyn plasmid, or 0.3 μg of Src plasmid, or 0.3 μg of PTPα plasmid; during the operation, the different DNA plasmids were first thoroughly mixed, and then the liposome transfection reagent was added. 3000 and P3000 should be separated, and Opti-MEM reduced serum medium was added as a buffer. After the DNA plasmid was added to the P3000 solution, it was combined with the 3000 solution and gently flicked with a finger to fully mix the DNA. After being coated with liposomes, it was incubated for 20 minutes, and then added to the cells and gently shaken for uniformity;

[0029] Step S23: After 8 h, the cells were washed once with PBS and replaced with DMEM low-glucose medium containing 10% FBS and without antibiotics, mainly because Lipofectamine 3000 solution was harmful to the cells.

[0030] Step S24: 24 h after transfection, digest the cells with Accutase and transfer them to a confocal dish coated with Fibronectin (10 μg / mL). Change the medium to DMEM low-glucose medium containing 1% FBS for starvation treatment. After the cells have attached for 24 h, start live cell imaging. The confocal dish coated with connexin needs to be prepared in advance and can be placed at 4°C and soaked in PBS for short-term storage.

[0031] Step S25, FRET microscopy imaging: The cell sample is placed in an incubation box to maintain a constant temperature of 5% CO2 and 37°C, and fixed on the microscope stage. The microscope is equipped with functions such as multi-point positioning, automatic focusing, and automatic switching of imaging filter channels. Fluorescence images are acquired using a 100x oil immersion lens. To reduce the photobleaching effect during the imaging process, the light source intensity is reduced to 1 / 32 to 1 / 64 before reaching the cell sample.

[0032] Furthermore, in step S25, the excitation channel filter and circular dichroism spectrometer parameters of the FRET probe donor and acceptor are both 430±15 nm and 455 nm, and the emission channel filter parameters are 480±20 nm and 530±15 nm, respectively.

[0033] The beneficial effects of the present invention are: the present invention is reasonably designed and has the following advantages:

[0034] (1) First, based on the FRET principle, a genetically encoded FRET biosensor was constructed using molecular cloning technology to measure the activity of Csk kinase. Among the three substrate peptides, the biosensor with EEEIYFFF as the substrate had better activity against Csk than the other two. This biosensor was selected and a DRM targeting motif containing the myristoylation and palmitoylation sites (glycine and cysteine) from Fyn and Lyn were fused to the N-terminus of the cytosolic Csk (Cyto-Csk) biosensor to construct Fyn-Csk and Lyn-Csk biosensors. At the same time, a polybasic geranyl motif (cysteine) adapted to Kras was fused to the carboxyl terminus of the Cyto-Csk biosensor to generate a Kras-Csk biosensor, which can be located in the general membrane area outside the DRM region.

[0035] (2) Fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide powerful tools for visualizing signaling molecules in living cells with high spatiotemporal resolution. They can specifically bind to Csk kinase in cells and visualize the dynamics of Csk kinase in cells, providing visualization tools for further exploring the mechanism of action of Csk in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] FIG1 is a schematic diagram of a biosensor according to the present invention;

[0038] FIG2 shows the conformational change of the biosensor of the present invention when tyrosine phosphorylation occurs on the substrate;

[0039] FIG3 is a diagram showing the characterization of different substrate polypeptides of the biosensor of the present invention and their submembrane localization;

[0040] FIG4 shows the FRET changes after the biosensor of the present invention is co-transfected with the CSK plasmid;

[0041] FIG5 is a characterization of the specificity of the biosensor of the present invention for Csk kinase;

[0042] FIG6 is a comparison of the FRET reactions produced by the biosensor of the present invention and multiple biosensors in ASM cells under PDGF induction;

[0043] FIG7 shows the regulation of CSK FRET reaction by co-transfection of PTPα plasmid into the biosensor of the present invention;

[0044] FIG8 shows the CSK FRET level of the biosensor of the present invention in tumor cells. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0046] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular also includes the plural. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0047] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] A biosensor for detecting intracellular Csk kinase activity is described. The biosensor sequentially fuses an N-terminal ECFP, the Src SH2 domain, a 15-amino acid flexible linker, a substrate peptide, and a C-terminal YPet. The substrate peptide is selected from the group consisting of the Src kinase C-terminal tail peptide FTSTEPQYQPGENL, the Fyn kinase C-terminal tail peptide FTATEPQYQPGENL, and the Csk-specific substrate peptide EEEIYFFF.

[0049] A detection method of the above-mentioned biosensor for detecting intracellular Csk kinase activity specifically comprises the following steps:

[0050] Step S1, molecular cloning construction, includes:

[0051] Step S11, PCR reaction system (100 μL) construction;

[0052] PCR reagent dosage

[0053] The reaction conditions are as follows:

[0054] Step S12, enzymatic digestion of the target fragment, comprises:

[0055] Amount of enzyme digestion reagent

[0056] After preparing ①②, place the plate in a 37℃ incubator for enzyme digestion for 3 hours.

[0057] After 3 h, remove ①, add CIAP (1 μL) (alkaline phosphatase) and 10× buffer (4.5 μL), and place in a 37°C incubator to continue enzyme digestion for 1 h;

[0058] Step S13, connecting the target fragment and the vector after enzyme digestion by T4 DNA ligase, comprising:

[0059] T4 DNA ligation reagent dosage

[0060] The target fragment and vector after enzyme digestion are connected by T4 DNA ligase; DNA and reagents are added and mixed according to the following ratio, and an empty vector is introduced as a control, and placed at room temperature for 1 hour.

[0061] Step S14, competent state transformation, includes:

[0062] Step S141: Take out a 50 μL tube of DH5α competent E. coli from a -80°C refrigerator and quickly freeze and thaw on ice;

[0063] Step S142: Add 0.5-2 μL of plasmid DNA to the competent E. coli and place on ice for 30 minutes;

[0064] Step S143, heat shock: place in a 42°C metal bath for 30-90 seconds;

[0065] Step S144, placing the EP tube on ice for 2 minutes;

[0066] Step S145: adding 1 mL of resistance-free LB medium;

[0067] Step S146: incubate at 37°C with shaking at 250 rpm for 1 h;

[0068] Step S147: Take out 250 μL of bacterial solution and apply it to the LB agar plate with the resistance corresponding to the plasmid DNA; if you want to increase the density of the bacteria on the plate, centrifuge the bacterial solution at 4000-8000 rpm for 1 minute, resuspend it in 250 μL of LB medium, and apply the entire bacterial solution to the plate;

[0069] Step S148: Place the culture plate upside down in a 37°C constant temperature incubator overnight;

[0070] Step S149: After the colonies grow, remove the agar plate; pick a single colony on a clean bench and place it in LB medium containing the corresponding resistance for culture and amplification; after 12 to 16 hours, extract the plasmid according to the corresponding steps;

[0071] Step S15, identification of the ligated plasmid, includes:

[0072] To verify the correctness of the extracted ligation product, first add the corresponding reagents according to the following ratio and mix thoroughly. Perform double digestion with SphⅠ and SacⅠ, using the vector plasmid as a control. Digest at 37°C for 2 hours, then separate by gel electrophoresis. Assuming the ligation product is correct, the gel electrophoresis plot will show two bands from top to bottom: 4300 bp (vector) and 300 bp (target fragment). The empty vector control will only have a 4300 bp (vector) band and no 300 bp (target fragment) band.

[0073] Enzyme digestion verification reagent dosage

[0074] Then, using the above molecular cloning steps, EcoRI and SalI restriction enzymes were selected to construct the FRET probe constructed above into the vector pCGGAS;

[0075] Membrane-targeted biosensors were constructed by PCR amplification of the biosensor and adding a Lyn tag (MGCIKSKRKDNLNDDGVDMKT) or a Fyn tag (MGCVQCKDKEATKLTEERDGSLNQ) to the N-terminus of the biosensor, which was then ligated into the pCAGGS vector via the EcoRI / SalI sites; a Kras tag (KKKKKKKSKTKCVI) was added to the C-terminus of the biosensor in the same manner;

[0076] Step S2, transfection of mammalian cells with FRET probe: transfecting the Csk FRET probe into cells using liposome transfection, culturing the cells in a 24-well cell culture plate, and transfecting or co-transfecting the Csk FRET probe, CSK plasmid, Fyn plasmid, Src plasmid, and PTPα plasmid, specifically comprising the following steps:

[0077] Step S21, culturing ASMC / MEF / Hela in 10% FBS DMEM low-glucose medium, with a small number of passages. When the cell density is 80-90%, the cells are seeded in a 24-well plate in advance. Preferably, the cell density is about 60% during transfection;

[0078] Step S22: According to the Lipofectamine 3000 liposome transfection method, each well was co-transfected with 1 μg of CskFRET plasmid DNA, 0.3 μg of CSK plasmid, or 0.3 μg of Fyn plasmid, or 0.3 μg of Src plasmid, or 0.3 μg of PTPα plasmid; during the operation, the different DNA plasmids were first thoroughly mixed, and then the liposome transfection reagent was added. 3000 and P3000 should be separated, and Opti-MEM reduced serum medium was added as a buffer. After the DNA plasmid was added to the P3000 solution, it was combined with the 3000 solution and gently flicked with a finger to fully mix the DNA. After being coated with liposomes, it was incubated for 20 minutes, and then added to the cells and gently shaken for uniformity;

[0079] Step S23: After 8 h, the cells were washed once with PBS and replaced with DMEM low-glucose medium containing 10% FBS and without antibiotics, mainly because Lipofectamine 3000 solution was harmful to the cells.

[0080] Step S24: 24 hours after transfection, digest the cells with Accutase and transfer them to a confocal dish coated with Fibronectin (10 μg / mL). Change the medium to DMEM low-glucose medium containing 1% FBS for starvation treatment. After the cells have attached for 24 hours, start live cell imaging. The confocal dish with the connexin needs to be prepared in advance and can be soaked in PBS at 4°C for short-term storage.

[0081] Step S25, FRET microscopy imaging: The cell sample was placed in an incubation box maintained at 5% CO2 and a constant temperature of 37°C (Zeiss) and fixed on the microscope stage. The microscope system was purchased from Zeiss and equipped with functions such as multi-point positioning, autofocus, and automatic switching of imaging filter channels. The excitation channel filters and circular dichroism spectrometer parameters for the FRET probe donor and acceptor were 430±15nm and 455nm, respectively, and the emission channel filters were 480±20nm and 530±15nm, respectively. Fluorescence images were acquired using a 100x oil immersion lens. The excitation light source was from an X-Cite Light Source (Lumen Dynamics). To reduce photobleaching during imaging, the light source intensity was reduced to 1 / 32 to 1 / 64 before reaching the cell sample.

[0082] Step S3, quantitative and statistical analysis of FRET image data: Data analysis was performed using the FRET image analysis software Fluocell 6.0.0, developed using the MATLAB software platform. Experimental analysis data were expressed as "mean + standard deviation" and analyzed using the statistical analysis software Origin 8.0 and GraphPad Prism 6.0. The differences between the data groups were compared using the t-test, and P < 0.05 indicated that the difference was statistically significant.

[0083] FIG1 is a schematic diagram of the principle of the biosensor of the present invention; FIG2 is a diagram showing the conformational change of the biosensor of the present invention when tyrosine phosphorylation occurs on a substrate.

[0084] Figure 3 shows (A) and (B) ECFP / FRET ratio images of ASMCs treated with PDGF. Multiple versions of the biosensor were constructed with different substrate peptides (A) or different membrane-targeting signal peptides (B). The color bars, from blue to red, represent the emission ratios (ECFP / FRET) of the biosensor from low to high, respectively. (C) and (D) show the time course of the ECFP / FRET emission ratios quantified in cells from (A) and (B). (E) Scatter plot with mean ± SD compares the basal and maximum ECFP / FRET ratios in cells compared with the time course shown in (B). (F) ECFP / FRET ratio images of ASMCs treated with PDGF. Multiple versions of the biosensor were constructed with different substrate peptides or Y / F mutants of different membrane-targeting signal peptides. (G) shows the time course of the ECFP / FRET emission ratios quantified in cells from (F). (H) Comparison of the normalized maximum ECFP / FRET ratios of the wild-type and Y / F mutant versions of the corresponding biosensors after PDGF stimulation in (A), (B), and (F). As shown in Figure 3, the biosensor of the present invention can specifically detect CSK kinase in cells, and CSK kinase is mainly present in the non-lipid raft region of the cell membrane; compared with the increase in the ECFP / FRET ratio of the Y / F mutant biosensor, it shows that the FRET response of the biosensor to CSK activation is due to the intramolecular interaction between the phosphorylated substrate and the SH2 domain.

[0085] Figure 4 shows (A) representative ECFP / FRET ratio images of ASMCs co-transfected with the KRas-Csk biosensor (1.0 μg DNA on a 24-well plate) and either vector or Csk mutants (0.3 μg DNA each) following PDGF stimulation. (B) average time course of ECFP / FRET ratios of the KRas-Csk biosensor in cells co-expressing Csk mutants or control vectors as shown in (A) when treated with PDGF. (C) scatter plot with mean ± SD comparing basal and maximum ECFP / FRET ratios in cells with the time course shown in (B). Figure 4 demonstrates that the biosensor of the present invention exhibits good specificity for CSK kinase.

[0086] Figure 5 shows (A) representative ECFP / FRET ratio images of PDGF-stimulated ASMCs (smooth muscle cells) co-transfected with the KRAS-Csk biosensor (1.0 μg DNA per 24-well plate) and either vector or Fyn and Src plasmids (0.3 μg DNA each). (B) Average time course of ECFP / FRET ratios of the KRAS-Csk biosensor in cells co-transfected with vector or Fyn and Src plasmids during PDGF treatment (mean ± standard error). (C) Scatter plot showing baseline and maximum ECFP / FRET ratios in cells co-transfected with vector or Fyn and Src plasmids during the time course shown in (B), along with mean ± standard deviation for comparison. (D) Representative ECFP / FRET ratio images of PDGF-stimulated ASMCs (smooth muscle cells) co-transfected with the KRAS-Csk biosensor (1.0 μg DNA per 24-well plate) and Csk-R107E (0.5 μg DNA each) along with vector or Fyn and Src plasmids (0.5 μg DNA each). (E) Average time course of ECFP / FRET ratios of the KRAS-Csk biosensor in co-transfected cells during PDGF treatment (mean ± standard deviation). (F) Scatter plot showing baseline and maximum ECFP / FRET ratios in cells over the time course shown in (E), along with mean ± standard deviation for comparison. As shown in Figure 5, the biosensor of the present invention exhibits superior specificity for CSK kinase relative to Src and Fyn kinases.

[0087] In Figure 6, (A) PDGF-stimulated ASMC cells were transfected with Csk-FRET, Src-FRET, Fyn-FRET, and FAK-FRET biosensors, and representative ECFP / FRET ratio images with membrane positioning are shown here. (B) The cells shown in (A) were quantified over the time course of the ECFP / FRET emission ratio. (C) The cells shown in (A) were quantified over the time course of FRET changes using ECFP / FRET. (D) A scatter plot (showing all individual data points and mean ± standard deviation) shows the quantification of FRET changes in ASMCs for 10 μg / mL PDGF biosensors. As shown in Figure 6, the biosensors of the present invention are less sensitive than Src-FRET, Fyn-FRET, and FAK-FRET biosensors under the same conditions.

[0088] Figure 7 shows (A) representative ECFP / FRET ratio images of PDGF-stimulated ASMCs (smooth muscle cells) co-transfected with the KRAS-Csk biosensor (1.0 μg DNA per 24-well plate) and cells containing either vector alone or the indicated PTPα mutants (1.0 μg DNA per plate). (B) representative ECFP / FRET ratio images of PDGF-stimulated ASMCs co-transfected with the KRAS-Csk biosensor (1.0 μg DNA per 24-well plate) and Csk-R107E (0.5 μg DNA per plate) and cells containing either vector alone or the indicated PTPα mutants (0.5 μg DNA per plate). (C) and (E) average time course of ECFP / FRET ratios of the KRAS-Csk biosensor in cells co-transfected with PTPα mutants or control vector during PDGF treatment (mean ± standard error). (D) and (F) Scatter plots showing the baseline and maximum ECFP / FRET ratios in cells during the time courses shown in (A) and (B), along with mean ± standard deviation for comparison. As shown in Figure 7 , the biosensor of the present invention demonstrates that PTPα has a certain inhibitory effect on Csk kinase activity.

[0089] Figure 8 (A) and (B) show representative ECFP / FRET ratio images of EGF-stimulated cells co-transfected with the KRas-Csk biosensor (using 1.0 μg of DNA in a 24-well plate). (C) and (E) show the average time course of ECFP / FRET ratios of the KRas-Csk biosensor when cells were stimulated with EGF (mean ± standard error). (D) and (F) show scatter plots showing the maximum ECFP / FRET ratios in the cells during the time courses shown in (C) and (E), along with the mean ± standard deviation for comparison. Figure 8 shows that the use of the biosensor of the present invention to detect CSK kinase activity in cancer cells demonstrates that the difference in Csk protein activity between cancer cells and non-cancerous cells is not significant.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biosensor for detecting the activity of Csk kinase in cells, characterized in that: The biosensor sequentially fuses and includes ECFP at the N-terminus, the SH2 domain of Src, a flexible linker with 15 amino acids, a substrate polypeptide, and YPet at the C-terminus; The substrate polypeptide is selected from one of the C-terminal tail peptide chains FTSTEPQYQPGENL of Src kinase, the C-terminal tail peptide chain FTATEPQYQPGENL of Fyn kinase, and the Csk-specific substrate peptide EEEIYFFF.

2. A detection method for the biosensor for detecting the activity of Csk kinase in cells according to claim 1, characterized in that: Specifically, it includes the following steps: Step S1, molecular cloning construction, including: Step S11, construction of the PCR reaction system; Step S12, digestion of the target fragment; Step S13, ligation of the digested target fragment and the vector with T4 DNA ligase; Step S14, competent cell transformation; Step S15, identification of the ligated plasmid; Step S2, transfection of the FRET probe into mammalian cells: Transfect the Csk FRET probe into cells by the liposome transfection method; Step S3, quantitative analysis and statistical analysis of FRET image data: Perform data analysis with the FRET image analysis software Fluocell 6.0.0 developed on the MATLAB software platform. The experimental analysis data is expressed as "mean + standard deviation", and analyzed with the statistical analysis software Origin 8.0 and GraphPad Prism 6.

0. The t-test is used to compare the differences between the data of each group, and P < 0.05 indicates that the difference is statistically significant.

3. The detection method for the biosensor for detecting the activity of Csk kinase in cells according to claim 2, characterized in that: The competent cell transformation in step S14 specifically includes the following steps: Step S141, Take out a tube of 50 μL of DH5α competent Escherichia coli from the -80°C refrigerator, Quickly freeze-thaw on ice; Step S142, Add 0.5 - 2 μL of plasmid DNA to the competent Escherichia coli and place it on ice for 30 min; Step S143, Heat shock: Place it in a 42°C metal bath for 30 - 90 s; Step S144, Place the EP tube on ice for 2 min; Step S145, Add 1 mL of antibiotic-free LB medium; Step S146, Incubate with shaking at 250 rpm at 37°C for 1 h; Step S147, Take out 250 μL of the bacterial solution and spread it on an LB agar plate with the corresponding resistance to the plasmid DNA. If you want to increase the density of the plated bacteria, centrifuge the bacterial solution at 4000 - 8000 rpm for 1 min, resuspend it with 250 μL of LB medium, and spread all the bacterial solution on the plate; Step S148, Invert the culture plate and incubate it overnight in a 37°C constant temperature incubator; Step S149, After colonies grow, take out the agar plate; Pick a single clone on the ultra-clean bench and place it in an LB medium containing the corresponding resistance for culture and amplification. Extract the plasmid according to the corresponding steps after 12 - 16 h.

4. The detection method for the biosensor for detecting the activity of Csk kinase in cells according to claim 2, characterized in that: In step S2, when transfecting the FRET probe into mammalian cells, culture the cells in a 24-well cell culture plate and transfect or co-transfect the Csk FRET probe, CSK plasmid, Fyn plasmid, Src plasmid, and PTPα plasmid. Specifically, it includes the following steps: Step S21: Culture ASMC / MEF / Hela in 10% FBS DMEM low-glucose medium with a small number of passages. When the cell density reaches 80-90%, inoculate the cells in a 24-well plate in advance. Preferably, the cell density at the time of transfection is about 60%. Step S22: According to the Lipofectamine 3000 liposome transfection method, co-transfect 1 μg of Csk FRET plasmid DNA, 0.3 μg of CSK plasmid, or 0.3 μg of Fyn plasmid, or 0.3 μg of Src plasmid, or 0.3 μg of PTPα plasmid into each well. When operating, first mix different DNA plasmids thoroughly, then add the liposome transfection reagent. Separate 3000 from P3000 and add Opti-MEM serum-free medium as a buffer. After adding the DNA plasmid to the P3000 solution, combine it with the 3000 solution, gently flick with fingers to make the DNA fully mixed and evenly coated with liposomes, incubate for 20 min, and then add it to the cells and gently shake to mix evenly. Step S23: After 8 h, wash the cells once with PBS and replace the medium with 10% FBS DMEM low-glucose medium without antibiotics. Step S24: 24 h after transfection, digest the cells with Accutase, transfer the cells to a confocal dish coated with Fibronectin (10 μg / mL), and change to 1% FBS DMEM low-glucose medium for starvation treatment. Start live cell imaging 24 h after the cells adhere. The confocal dish coated with the connecting protein needs to be prepared in advance and can be stored short-term by soaking in PBS at 4°C. Step S25: Perform FRET microscope imaging. Place the cell sample in an incubation box to maintain a constant temperature of 5% CO2 and 37°C, and fix it on the microscope stage. The microscope is equipped with functions such as multi-point positioning, automatic focusing, and automatic switching of imaging filter channels. Use a 100× oil immersion lens for fluorescence image acquisition. To reduce the photobleaching effect during imaging, the light source intensity is reduced to 1 / 32-1 / 64 of the original level before reaching the cell sample.

5. The detection method for the biosensor for detecting the activity of Csk kinase in cells according to claim 4, characterized in that: In the above step S25, the excitation channel filter and circular dichroic spectroscope parameters of the FRET probe donor and acceptor are commonly 430 ± 15 nm and 455 nm, and the emission channel filter parameters are 480 ± 20 nm and 530 ± 15 nm respectively.

Images