Fluorescent probe of phosphatidylinositol-3, 4, 5-triphosphoric acid, plasmid, preparation and kit

Abstract

The invention discloses a fluorescent probe of phosphatidylinositol-3, 4, 5-triphosphoric acid, a plasmid, a preparation method and application, relates to the technical field of biology, and aims to solve the problems of low affinity, low specificity and low sensitivity of the existing fluorescent probe of phosphatidylinositol-3, 4, 5-triphosphoric acid. According to the fluorescent probe, two sections of PH-TH structural domain coding sequences of human-derived Btk connected by a first connecting peptide are utilized, a key binding structural domain is subjected to series dimerization, the affinity of the key binding structural domain is amplified by multiple times, the specificity and the sensitivity are enhanced, the structural domain coding sequences are screened and simplified, the cytotoxicity is reduced, and the stability of the probe is improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a fluorescent probe, a plasmid and a kit of phosphatidylinositol-3,4,5-triphosphate. Background Art

[0002] PtdIns(3,4,5)P3 is a phosphatidylinositol molecule located on the cell membrane. It has glyceraldehyde as its backbone, C1- and C2- are connected to the acyl group, C3- is connected to the inositol head group, and C3-, C4-, and C5- of the inositol head group are all phosphorylated. PtdIns(3,4,5)P3 regulates multiple signaling pathways including Akt, affecting cell growth, proliferation, energy metabolism, and cell cycle. Akt activation depends on the recruitment of PtdIns(3,4,5)P3. When the level of PtdIns(3,4,5)P3 is abnormally elevated due to type I PI3K activating mutations or PTEN loss of function, excessive PtdIns(3,4,5)P3 will recruit too much Akt, further promoting cancer cell proliferation and invasion, and driving tumor development.

[0003] There are seven phosphatidylinositol molecules in cells, among which PtdIns(3,4)P2, PtdIns(3,5)P2, and PtdIns(4,5)P2 are similar in structure to PtdIns(3,4,5)P3. In addition, due to its small size and fast movement, PtdIns(3,4,5)P3 has the lowest relative content among the seven phosphatidylinositols, so its detection and imaging in living cells is still quite challenging. Currently, the detection of PtdIns(3,4,5)P3 in living cells mainly uses the binding domains of different proteins, such as Btk, Grp1 (general receptor of phosphoinositides 1, Grp1), ARNO (Arf nucleotide-binding site opener, ARNO) and the PH domain of Akt, but these methods all have limitations. For example, the affinity and specificity of PH-Akt binding to PtdIns(3,4,5)P3 are not high; although PH-Btk has a higher affinity, its sensitivity is insufficient; and the binding specificity of PH-Grp1 and PH-ARNO is also not high.

[0004] Therefore, there is an urgent need for a PtdIns(3,4,5)P3 probe that can achieve high specificity, high affinity, and high sensitivity imaging. Summary of the invention

[0005] In view of the above analysis, the present invention aims to provide a fluorescent probe, plasmid and kit of phosphatidylinositol-3,4,5-triphosphate to solve the problems of low affinity, low specificity and insufficient sensitivity in the prior art when detecting PtdIns(3,4,5)P3.

[0006] The purpose of the present invention is mainly achieved through the following technical solutions:

[0007] In one aspect, the present invention provides a fluorescent probe of phosphatidylinositol-3,4,5-triphosphate, which comprises a sequence of the fluorescent probe as shown in SEQ ID NO.1.

[0008] Furthermore, the fluorescent probe includes a first human Btk PH-TH domain coding sequence and a second human Btk PH-TH domain coding sequence connected by a first connecting peptide.

[0009] Furthermore, the fluorescent probe also includes a fluorescent protein coding sequence and a Flag tag coding sequence which are sequentially connected to the C-terminus of the PH-TH domain coding sequence of the second human Btk through a second connecting peptide.

[0010] Furthermore, the fluorescent protein is mNeonGreen fluorescent protein.

[0011] Furthermore, the fluorescent probe also includes a nuclear export peptide connected to the N-terminus of the PH-TH domain of the first human Btk.

[0012] Furthermore, the first connecting peptide is a flexible polypeptide chain consisting of valine, aspartic acid, serine and glycine.

[0013] Furthermore, the second connecting peptide is an amino acid sequence including two sections of flexible connecting peptides, the second connecting peptide includes 4 to 6 polypeptides, and the sequence of the polypeptide is GGGGS.

[0014] The second aspect of the present invention also provides a plasmid, which comprises a nucleotide sequence encoding the fluorescent probe of the above-mentioned phosphatidylinositol-3,4,5-triphosphate.

[0015] The third aspect of the present invention also provides a method for constructing the above-mentioned plasmid, which comprises:

[0016] Step 1, obtaining the PH-TH domain coding sequence of the first human Btk, the first connecting peptide sequence, the PH-TH domain coding sequence of the second human Btk, the second connecting peptide sequence, the mNeonGreen fluorescent protein coding sequence and the Flag tag coding sequence by PCR amplification;

[0017] Step 2: Connect the sequence obtained in step 1 into the pEGFP-C1 vector carrying the nuclear export peptide coding sequence through homologous recombination to form a plasmid structure.

[0018] The fourth invention of the present invention also provides a kit for detecting phosphatidylinositol-3,4,5-triphosphate, which comprises the probe described above.

[0019] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0020] (1) The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate provided by the present invention screens and prunes the key binding domain residues of the PH-TH domain of human Btk, thereby reducing the volume and the toxicity of the probe to cells; at the same time, the key binding domains are dimerized in series using the first connecting peptide, thereby amplifying the specificity, affinity and sensitivity of the fluorescent probe of phosphatidylinositol-3,4,5-triphosphate to PtdIns(3,4,5)P3 by several times.

[0021] (2) The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate provided by the present invention connects the fluorescent protein to the C-terminus of the fluorescent probe domain, and the N-terminus of the PH structure of Btk on the fluorescent probe is completely used to bind to PtdIns(3,4,5)P3, thereby avoiding the influence of the expression of the fluorescent protein on the binding of its N-terminal domain to PtdIns(3,4,5)P3. In addition, compared with binding to the N-terminus, the fluorescent protein is bound to the C-terminus of the probe, and the probe has better stability and reduced degradation risk.

[0022] (3) The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate provided by the present invention adds a more flexible connecting peptide structure in the probe design to reduce the toxicity of the probe.

[0023] Other features and advantages of the present invention will be described in the following description, and part of them will become obvious from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the written description and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are only for the purpose of illustrating the particular invention and are not to be considered as limiting the invention. The same reference symbols denote the same components throughout the accompanying drawings.

[0025] Figure 1 A plasmid expression vector map of the fluorescent probe described in the present invention;

[0026] Figure 2The imaging difference diagrams of the PtdIns(3,4,5)P3 probe of the embodiment of the present invention and the comparative example GFP-PH(Btk) probe in different cells; (A) is mesenchymal stem cells (MSCs), and (B) is embryonic stem cells (ESCs); the distribution diagram of the probe on the cell membrane is a fluorescence photograph obtained by 488nm laser irradiation under a spinning disk confocal microscope, and the bar graph is a statistical fluorescence intensity difference when the two probes are expressed in the same cell; scale bar = 5μm;

[0027] Figure 3 It is a statistical graph of the fluorescence intensity of the PtdIns (3,4,5) P3 probe of the embodiment of the present invention, the GFP-PH (Btk) probe of the comparative example and the mutant probe in ESCs;

[0028] Figure 4 The fluorescence intensity change curves of the selectivity and sensitivity verification test of the PtdIns (3,4,5) P3 probe of the embodiment of the present invention and the GFP-PH (Btk) probe of the comparative example in ESCs cells;

[0029] Figure 5 This is a sensitivity quantification diagram of the PtdIns (3,4,5) P3 probe of the present invention and the comparative example GFP-PH (Btk) probe in ESCs cells;

[0030] Figure 6 This is a quantitative bar graph of the survival rates of the PtdIns(3,4,5)P3 probe of the embodiment of the present invention, the GFP-PH(Btk) probe of the comparative example, the GFP-2xPH(Btk) probe and the GFP-3xPH(Btk) probe when expressed in ESCs cells. DETAILED DESCRIPTION

[0031] The present invention is further illustrated by examples below in conjunction with the accompanying drawings, but is not intended to limit the present invention. The specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, and materials identical or similar to the types, models, qualities, properties or functions of the following reagents and instruments can be used to implement the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0032] The present invention provides a fluorescent probe of phosphatidylinositol-3,4,5-triphosphate, which comprises a sequence of the fluorescent probe as shown in SEQ ID NO.1.

[0033] The probe shown in the present invention screens and prunes the key binding domain residues of the PH-TH domain of human Btk, reduces the volume, and reduces the toxicity of the probe to cells; at the same time, the key binding domains are dimerized in series using the first connecting peptide, thereby amplifying the specificity, affinity and sensitivity of the fluorescent probe of phosphatidylinositol-3,4,5-triphosphate to PtdIns(3,4,5)P3 by several times.

[0034] In the above scheme, the fluorescent probe includes a first human Btk PH-TH domain coding sequence and a second human Btk PH-TH domain coding sequence connected by a first connecting peptide.

[0035] The PH-TH domain coding sequence of the first human Btk is from position 2 to position 170 in the PH-TH domain of the first human Btk; for example, the PH-TH domain coding sequence of the first human Btk is from position 2 to position 169 in the PH-TH domain of the first human Btk; or, the PH-TH domain coding sequence of the first human Btk is from position 2 to position 168 in the PH-TH domain of the first human Btk; preferably, the PH-TH domain coding sequence of the first human Btk is from position 2 to position 167 in the PH-TH domain of the first human Btk; more preferably, the PH-TH domain coding sequence of the first human Btk is from position 2 to position 166 in the PH-TH domain of the first human Btk.

[0036] The PH-TH domain coding sequence of the second human Btk is positions 2 to 170 in the PH-TH domain of the first human Btk; for example, the PH-TH domain coding sequence of the second human Btk is positions 2 to 169 in the PH-TH domain of the first human Btk; or, the PH-TH domain coding sequence of the second human Btk is positions 2 to 168 in the PH-TH domain of the first human Btk; preferably, the PH-TH domain coding sequence of the second human Btk is positions 2 to 167 in the PH-TH domain of the first human Btk; more preferably, the PH-TH domain coding sequence of the second human Btk is positions 2 to 166 in the PH-TH domain of the first human Btk.

[0037] In the above scheme, the fluorescent probe also includes a fluorescent protein coding sequence and a Flag tag coding sequence which are sequentially connected to the C-terminus of the PH-TH domain coding sequence of the second human Btk through a second connecting peptide; the fluorescent protein is mNeonGreen fluorescent protein.

[0038] The fluorescent probe also includes a nuclear export peptide connected to the N-terminus of the PH-TH domain of the first human Btk.

[0039] In the above scheme, the first connecting peptide is a flexible polypeptide chain composed of valine, aspartic acid, serine and glycine.

[0040] The second connecting peptide is an amino acid sequence including two sections of flexible connecting peptides. The second connecting peptide includes 4 to 6 polypeptides, and the sequence of the polypeptide is GGGGS.

[0041] The present invention also provides a plasmid, such as Figure 1 As shown, it includes a nucleotide sequence encoding the fluorescent probe of the above-mentioned phosphatidylinositol-3,4,5-triphosphate.

[0042] The present invention also provides a method for constructing the above-mentioned plasmid, which comprises:

[0043] Step 1, obtaining the PH-TH domain coding sequence of the first human Btk, the first connecting peptide sequence, the PH-TH domain coding sequence of the second human Btk, the second connecting peptide sequence, the mNeonGreen fluorescent protein coding sequence and the Flag tag coding sequence by PCR amplification;

[0044] Step 2: Connect the sequence obtained in step 1 into the pEGFP-C1 vector carrying the nuclear export peptide coding sequence through homologous recombination to form a plasmid structure.

[0045] The present invention also provides a kit for detecting phosphatidylinositol-3,4,5-triphosphate, which comprises the above-mentioned probe.

[0046] 1. Preparation of the fluorescent probe of phosphatidylinositol-3,4,5-triphosphate (hereinafter referred to as the PtdIns(3,4,5)P3 probe)

[0047] 1) Design reference:

[0048] PI (3,4,5) P3 recognizes the binding domain coding sequence, using Addgene (Plasmid # 86635) as a template, and using Premier 5.0 software and NCBI BLAST analysis to design primers, and obtain primers F1 and R1 for the PH-TH domain coding sequence of the first human Btk, and primers F2 and R2 for the PH-TH domain coding sequence of the second human Btk. The specific primer information is as follows:

[0049] F1:5'-gagctggatgagGCACCGGTCGCCGCAGTGATTCTGGAGAG-3';

[0050] R1:5'-tcgacaccacccgatcccccTTGGCAGCCCATAGCATTTT-3';

[0051] F2:5'-ggggatcgggtggtgtcgacGCCGCATGATTCTGGAGAG-3';

[0052] R2:5'-CCACCGGAACCACCTCCTCCTTGGCAGCCCATAGCATTTT-3'.

[0053] The mNeonGreen fluorescent protein coding sequence and the Flag tag protein coding sequence were obtained using Addgene (Plasmid #98882) as a template, and the primers were designed using Premier 5.0 software and NCBI BLAST analysis to obtain primers F3 and R3 for the mNeonGreen fluorescent protein coding sequence and the Flag tag protein coding sequence;

[0054] F3:5'-GGAGGAGGTGGTTCCGGTGGAGGTGGATCTgtgagcaagggcg aggagga-3',

[0055] R3:5'-GGATCCCGGGCCCGCGGTTACTTGTCGTCGTCGTCCTTGTAGTCcttgtacagctcgtccatgc-3'.

[0056] 2) Preparation of fluorescent probes

[0057] Target fragment amplification PCR, the reaction system of PCR amplification is shown in the following table:

[0058] DNA template 100ng Forward primer (10 μM) 1μL Reverse primer (10 μM) 1μL 2X KAPA HiFi HotStart ReadyMix 12.5μL Distilled water (ddH2O) Up to 25μL Total reaction mentions 25μL

[0059] The PCR amplification program was as follows: denaturation at 95°C for 5 min, 30 cycles of 98°C for 20 s, 65°C for 15 s, and 72°C for 60 s (1000 bp / min)), and extension at 72°C for 5 min.

[0060] The PH-TH domain coding sequence of the first human Btk, the PH-TH domain coding sequence of the second human Btk, the mNeonGreen fluorescent protein coding sequence and the Flag tag protein coding sequence were obtained.

[0061] The first connecting peptide is a flexible polypeptide chain consisting of valine, aspartic acid, serine and glycine;

[0062] The second connecting peptide is composed of two flexible connecting peptides to form a soft, linear, and easily bendable amino acid sequence. The second connecting peptide includes 4 to 6 polypeptides, and the sequence of the polypeptide is GGGGS.

[0063] The above sequence was connected into the pEGFP-C1 vector with NES (nuclear export peptide) coding sequence by homologous recombination to form the plasmid structure of phosphatidylinositol-3,4,5-triphosphate fluorescent probe.

[0064] 2. Comparative Example A plasmid containing a PH-Btk region was used as a comparative example GFP-PH (Btk) probe, which was from Addgene (Plasmid#86635).

[0065] 3. Cultivation of mesenchymal stem cells MSCs and embryonic stem cells ESCs.

[0066] MSCs cell lines were cultured at 37°C, 5% CO 2 CO 2 The MSCs cell line was cultured in a DMEM medium containing 2% fetal bovine serum and 1% double antibody. When the cells entered the logarithmic phase, the cell layer was treated with protease for 4 minutes, and then the reaction was terminated with a medium containing complete nutrients. After that, a predetermined amount of cells were taken from the treated cell suspension and inoculated into a dedicated confocal imaging dish and placed in a CO 2 Culture in an incubator for 24-48 hours.

[0067] ESCs were cultured at 37°C with 5% CO 2 , 5% O 2 CO 2 The ESCs cell line was cultured in E8 medium and 1% double-antibody DMEM culture medium. When the cells entered the logarithmic phase, the cell layer was treated with protease for 4 minutes, and then the reaction was terminated with a culture medium containing complete nutrients. After that, a predetermined amount of cells were taken from the treated cell suspension and inoculated into a dedicated confocal imaging dish and placed in a CO 2 Culture in an incubator for 24-48 hours.

[0068] 4. Detect the fluorescent probe of phosphatidylinositol-3,4,5-triphosphate in the example and the GFP-PH (Btk) probe in the comparative example in MSCs cells and ESCs cells respectively.

[0069] The comparative example probe and the example probe were expressed in different MSCs and ESCs cells by liposome transfection, and imaging was performed after 8-12 hours. Figure 2As shown, (A) is mesenchymal stem cells (MSCs), and (B) is embryonic stem cells (ESCs). The distribution diagram of the probe on the cell membrane is a fluorescent photo obtained by 488nm laser irradiation under a rotating disk confocal microscope, and the bar graph is a statistical difference in fluorescence intensity when the two probes are expressed in the same cell. Scale bar = 10μm. It can be seen that compared with the comparative example GFP-PH (Btk) probe, the fluorescent probe of the embodiment phosphatidylinositol-3,4,5-triphosphate has a stronger ability to display the distribution of PtdIns (3,4,5) P3.

[0070] 5. Specificity verification.

[0071] When ESCs cells grow to the logarithmic phase, the cells are digested with protease for 4 minutes. After termination with complete medium, a certain number of cells are inoculated into a confocal imaging dish and placed in CO 2 After culturing in an incubator for 24-48 hours. The comparative example GFP-PH (Btk) probe, the example PtdIns (3,4,5) P3 probe and the mutant probe were expressed in ESCs cells by liposome transfection, and imaging and analysis were performed 8-12 hours later. Among them, the mutant probe is a key binding site mutant of the example PtdIns (3,4,5) P3 probe, which introduces the R28C point mutation into the key site of the example PtdIns (3,4,5) P3 probe binding to PtdIns (3,4,5) P3.

[0072] The quantitative analysis results are as follows Figure 3 As shown in the figure, compared with the PtdIns(3,4,5)P3 probe of the embodiment, the fluorescence intensity of the mutant probe on the cell membrane is significantly reduced, thereby confirming the targeting of the PtdIns(3,4,5)P3 probe of the embodiment to the PtdIns(3,4,5)P3 molecule on the cell membrane. This phenomenon shows that the fluorescence signal intensity of the PtdIns(3,4,5)P3 probe of the embodiment on the cell membrane directly reflects the local concentration of PtdIns(3,4,5)P3. In addition, as Figure 4 As shown, compared with the comparative example GFP-PH (Btk) probe and the example PtdIns (3,4,5) P3 probe, the novel PtdIns (3,4,5) P3 probe exhibits higher specificity in indicating the distribution of PtdIns (3,4,5) P3 in cells.

[0073] 6. Selectivity and sensitivity verification.

[0074] When ESCs cells grow to the logarithmic phase, use protease to digest the cells for 4 minutes. After the digestion, take a certain number of cells and inoculate them into a confocal imaging dish and place them in CO 2After culturing in an incubator for 24-48 hours, the comparative example GFP-PH (Btk) probe and the example PtdIns (3,4,5) P3 probe were expressed in ESCs cells by liposome transfection, and imaging was performed 8-12 hours later. At 2 minutes after the start of shooting, an agonist (A) that promotes the production of PtdIns (3,4,5) P3 or an inhibitor (B) that inhibits the production of PtdIns (3,4,5) P3 were used to treat the cells, and then the changes in the fluorescence intensity of each probe were quantitatively measured to evaluate its response to changes in the level of PtdIns (3,4,5) P3.

[0075] Results Figure 4 and Figure 5 When the cell membrane PtdIns(3,4,5)P3 content increases or decreases, the PtdIns(3,4,5)P3 probe of the embodiment responds more sensitively to the cell membrane PtdIns(3,4,5)P3, and the sensitivity is nearly 4 times higher than that of the comparative example GFP-PH(Btk) probe. Figure 5 The figure is a bar graph quantifying the sensitivity difference between the example PtdIns(3,4,5)P3 probe and the comparative example GFP-PH(Btk) probe.

[0076] 7. Low toxicity test

[0077] When ESCs reached the logarithmic growth phase, the cell layer was treated with protease for 4 minutes, and then complete medium was added to terminate the reaction. Next, a predetermined amount of cells were taken out and seeded into a confocal imaging dish, which was then placed in a CO 2 The cells were cultured in an incubator for 24-48 hours. The comparative GFP-PH (Btk) probe, GFP-2xPH (Btk) probe, GFP-3xPH (Btk) probe, and the example PtdIns (3,4,5) P3 probe were expressed in ESCs by liposome-mediated transfection technology. 12 hours after transfection, the Cell Counting Kit-8 (CCK-8) method was used to evaluate the cytotoxicity of the probes to ESCs under different transfection conditions. Figure 6 As shown, the results show that, similar to the comparative GFP-PH (Btk) probe, the novel PtdIns (3,4,5) P3 probe exhibits extremely low cytotoxicity to ESCs. The comparative GFP-PH (Btk) probe exhibits low cytotoxicity due to its smaller molecular size; after structural optimization, the PtdIns (3,4,5) P3 probe of the embodiment also achieves extremely low cytotoxicity based on tandem dimerization of the binding domain; relatively speaking, due to their larger molecular size, the GFP-2xPH (Btk) probe and the GFP-3xPH (Btk) probe exhibit relatively high cytotoxicity.

[0078] The PtdIns(3,4,5)P3 probe of the embodiment has lower cytotoxicity than the GFP-2xPH(Btk) probe of similar molecular size, and is similar in toxicity to the comparative GFP-PH(Btk) probe of smaller molecular size. In addition, the comparative GFP-PH(Btk) probe has better PtdIns(3,4,5)P3 affinity, higher binding specificity and higher sensitivity.

[0079] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A fluorescent probe of phosphatidylinositol-3,4,5-triphosphate, characterized in that: The sequence including the fluorescent probe is shown in SEQ ID NO.

1.

2. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 1, characterized in that: The fluorescent probe includes a first human Btk PH-TH domain encoding sequence and a second human Btk PH-TH domain encoding sequence connected by a first connecting peptide.

3. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 2, characterized in that: The fluorescent probe also includes a fluorescent protein coding sequence and a Flag tag coding sequence which are sequentially connected to the C-terminus of the PH-TH domain coding sequence of the second human Btk through a second connecting peptide.

4. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 3, characterized in that: The fluorescent protein is mNeonGreen fluorescent protein.

5. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 2, characterized in that: The fluorescent probe also includes a nuclear export peptide connected to the N-terminus of the PH-TH domain of the first human Btk.

6. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 2, characterized in that: The first connecting peptide is a flexible polypeptide chain consisting of valine, aspartic acid, serine and glycine.

7. The fluorescent probe of phosphatidylinositol-3,4,5-triphosphate according to claim 3, characterized in that: The second connecting peptide is an amino acid sequence including two sections of flexible connecting peptides, the second connecting peptide includes 4 to 6 polypeptides, and the sequence of the second connecting peptide is GGGGS.

8. A plasmid, characterized in that It comprises a nucleotide sequence encoding the fluorescent probe according to any one of claims 1 to 7.

9. A method for constructing the plasmid according to claim 8, characterized in that: include: Step 1, obtaining the PH-TH domain coding sequence of the first human Btk, the first connecting peptide sequence, the PH-TH domain coding sequence of the second human Btk, the second connecting peptide sequence, the mNeonGreen fluorescent protein coding sequence and the Flag tag coding sequence by PCR amplification; Step 2: Connect the sequence obtained in step 1 into the pEGFP-C1 vector carrying the nuclear export peptide coding sequence through homologous recombination to form a plasmid structure.

10. A kit for detecting phosphatidylinositol-3,4,5-triphosphate, characterized in that: The fluorescent probe comprises any one of claims 1 to 7.

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