A fluorescent probe for spermidine and a preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and provides a spermidine fluorescent probe, a preparation method and application thereof. The spermidine fluorescent probe comprises a spermidine-sensitive polypeptide and an optically active polypeptide. The spermidine fluorescent probe has the advantages of relatively small protein molecular weight, easy maturation, large fluorescence dynamic change, good specificity, expression in cells through a gene operation method, and high specificity, high throughput, real-time and quantitative dynamic detection of spermidine in and out of cells.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probe technology, and in particular to a spermidine fluorescent probe, its preparation method, and its application. Background Technology

[0002] Spermidine is an organic cationic compound with three amino groups, widely found in plants, animals, and microorganisms. In recent years, it has become a research hotspot due to its multiple biological activities in areas such as anti-aging, metabolic regulation, and neuroprotection. As a core intermediate in polyamine metabolism, spermidine can be synthesized de novo using arginine as a precursor, and can also be obtained through dietary supplementation from spermidine-rich foods such as wheat germ, mushrooms, and cheese. Artificial supplementation with spermidine has shown effects in various biological models, including extending lifespan, improving cognitive function, maintaining cell stemness, and protecting cardiac function.

[0003] Currently, quantitative detection of spermidine mainly relies on mass spectrometry, which requires sample pretreatment such as lysis, extraction, lyophilization, and derivatization of cells and tissues. This process is complex and time-consuming, and can only collect metabolite concentration information at a single time point, failing to provide dynamic monitoring of concentration. Because the precise volume measurement and calculation of cells and organelles are difficult, mass spectrometry methods struggle to perform in-situ quantitative calculations of metabolite concentrations within cells and even subcellular compartments; relative quantification is only possible using cell count or protein content as a reference. Therefore, there is an urgent need to develop new detection methods to achieve highly specific, high spatiotemporal resolution, and high-throughput quantitative detection of spermidine both intracellularly and in vitro. Summary of the Invention

[0004] The purpose of this invention is to provide a spermidine fluorescent probe, its preparation method and application, which has multiple advantages such as high spatiotemporal resolution, high throughput and precise quantification.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a spermidine fluorescent probe, comprising a spermidine-sensitive polypeptide and an optically active polypeptide.

[0006] Preferably, the spermidine-sensitive polypeptide is any one of spermidine-binding protein, spermidine-binding protein functional variant 1, spermidine-binding protein functional variant 2, and spermidine-binding protein functional variant 3; the spermidine-sensitive polypeptide is derived from Pseudomonas aeruginosa; the amino acid sequence of the spermidine-binding protein is shown in SEQ ID NO:1; The spermidine-binding protein functional variant 1 is obtained by mutation of the spermidine-binding protein; the mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion. The mutation sites are any 1 to 5 of the following: T35, D36, P81, T134, V141, V147, P152, A157, E161, D180, E181, A185, N195, H197, K200, K203, F219, K223, Y239, D242, N269, W271, D273, P297, A301, Y309, L319, and Y331.

[0007] Preferably, the mutations are any combination of the following: F219 and N269; P81, F219 and N269; V141, F219 and N269; F219, K223 and N269; E181, F219, K223 and N269; T134, E161, F219 and N269; F219, N269, L319 and Y331; D180, F219, N269, L319 and Y331; T134, E161, D180, F219 and N269; T134, F219, N269 and L319; K200, F219, N269 and L319; E161, A185, F219 and N269; The mutations are as follows: P81 mutates to Q, T134 mutates to I, E161 mutates to I, D180 mutates to A, E181 mutates to A, A185 mutates to M, K200 mutates to I, F219 mutates to W or D, K223 mutates to Q, N269 mutates to G or P, L319 mutates to D or E, and Y331 mutates to G.

[0008] Preferably, the mutations are any combination of the following: F219D and N269G; F219W and N269G; P81Q, F219D and N269G; F219W, K223Q and N269G; E181A, F219W, K223Q and N269G; T134I, E161I, F219W and N269G; F219W, N269G, L319D and Y331G; D180A, F219W, N269G, L319D and Y331G; T134I, E161I, D180A, F219W and N269G; T134I, F219W, N269G and L319D; K200I, F219W, N269G and L319D; E161I, A185M, F219W and N269G; V141L, F219W and N269G.

[0009] Preferably, the spermidine-binding protein functional variant 2 is a truncated variant of amino acids 28 to 362 of the amino acid sequence of spermidine-binding protein functional variant 1. The spermidine-binding protein functional variant 3 is a nucleotide sequence that has ≥70% sequence similarity to spermidine-binding protein functional variant 1.

[0010] Preferably, the optically active polypeptide is located between residues 67-72, 218-224, or 337-342 of the spermidine-sensitive polypeptide; The optically active polypeptide is any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any one of the sequences containing 1 to 6 mutations at any of the Y1, N2, D4, K22, I27, F59, K62, S130, Y139, F199, and N205 sites in SEQ ID NO:2; The mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion.

[0011] Preferably, the mutation is any one or more of the following: Y1G, N2G, D4H, K22W, I27V, F59Y, K62A, S130R, Y139N, F199T and N205F; The optically active polypeptide is located at one or more of the following sites on the spermidine-sensitive polypeptide: 67 / 68, 67 / 69, 67 / 70, 67 / 71, 67 / 72, 68 / 69, 68 / 70, 68 / 71, 68 / 72, 69 / 70, 69 / 71, 69 / 72, 70 / 71, 70 / 72, 71 / 72, 218 / 219, 218 / 220, 218 / 221, 218 / 222, 218 / 223, 218 / 224, 219 / 220, 219 / 221, 219 / 222, 219 / 223. 219 / 224, 220 / 221, 220 / 222, 220 / 223, 220 / 224, 221 / 222, 221 / 223, 221 / 224, 222 / 223, 222 / 224, 223 / 224, 337 / 338, 337 / 339, 337 / 340, 337 / 341, 337 / 342, 338 / 339, 338 / 340, 338 / 341, 338 / 342, 339 / 340, 339 / 341, 339 / 342, 340 / 341, 341 / 342.

[0012] Preferably, the amino acid sequence of the spermidine fluorescent probe is any one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0013] The present invention also provides a nucleotide sequence encoding the spermidine fluorescent probe.

[0014] The present invention also provides the application of the spermidine fluorescent probe or the nucleotide sequence encoding the spermidine fluorescent probe in the localization or quantitative detection of spermidine.

[0015] In one or more embodiments, the cpVenus variant has the sequence shown in SEQ ID NO:2 and has mutations at one, two, three, four, five, or six of the following sites: Y1, N2, D4, K22, I27, S130, Y139, F199, N205, where the mutations include amino acid modifications, substitutions, or deletions. In one or more embodiments, the mutations in the cpVenus variant are Y1G, N2G, and D4H.

[0016] In one or more embodiments, the fluorescent probe comprises any of the amino acid sequences SEQ ID NO:10-17 or variations thereof. In one or more embodiments, the fluorescent probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-18. Preferably, the fluorescent probe provided by the present invention comprises a sequence substantially similar to or identical to any of the amino acid sequences SEQ ID NO:10-18.

[0017] Another aspect of the present invention provides a fusion polypeptide comprising the fluorescent probe described herein and other polypeptides. In some embodiments, the fluorescent probe described herein further comprises other polypeptides fused thereto. These other polypeptides do not affect the properties of the fluorescent probe. In some embodiments, the other polypeptides are located at the N-terminus and / or C-terminus of the fluorescent probe. In some embodiments, the other polypeptides include polypeptides for targeting the fluorescent probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the fluorescent probe and other polypeptides in the fusion polypeptide described herein.

[0018] Another aspect of the present invention also provides a nucleic acid molecule comprising: (a) The coding sequence of the polypeptide or probe described in any embodiment of this document, or (b) The complementary sequence of (a), or (c) A fragment of (a) or (b). The fragment is a primer.

[0019] In one or more embodiments, the nucleic acid sequence comprises an amino acid sequence encoding any of the amino acid sequences shown in SEQ ID NO:10-18.

[0020] Preferably, the nucleic acid sequence comprises any of the nucleotide sequences SEQ ID NO:10-18 or a variant thereof.

[0021] More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70%, or 50% identity with any of the nucleotide sequences SEQ ID NO:10-18; or comprises a nucleotide sequence that is substantially similar to or identical to any of the nucleotide sequences SEQ ID NO:10-18.

[0022] The present invention also relates to complementary sequences or variants of the above-mentioned nucleic acid sequences, which may contain nucleic acid sequences or complementary sequences of fragments, analogs, derivatives, soluble fragments and variants encoding the fluorescent probes or fusion proteins of the present invention.

[0023] This invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes the fluorescent probe or fusion polypeptide described herein.

[0024] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.

[0025] In one or more embodiments, the nucleic acid molecule is operatively linked to an expression control sequence.

[0026] In some implementations, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors, and viral vectors.

[0027] The present invention also provides a host cell comprising: (1). Expressing the fluorescent probe or fusion polypeptide described in any embodiment of the present invention; (2) A nucleic acid molecule comprising any embodiment of the present invention; or (3) A nucleic acid construct comprising any embodiment of the present invention. The host cell is preferably Escherichia coli.

[0028] Another aspect of the present invention provides a spermidine detection kit, comprising the fluorescent probes described herein or fusion peptides or polynucleotides or fluorescent probes prepared as described herein.

[0029] In one or more embodiments, the kit further comprises one or more reagents selected from the following: buffer solution, culture medium, spermidine standard.

[0030] The present invention provides a method for preparing the fluorescent probe described herein, comprising: providing a host cell expressing the fluorescent probe or fusion polypeptide described herein, culturing the host cell under conditions of expression of the fluorescent probe or fusion polypeptide, and isolating the fluorescent probe or fusion polypeptide.

[0031] In one or more embodiments, the method for preparing the spermidine fluorescent probe or fusion peptide described herein includes the following steps: 1) The expression vector encoding the spermidine fluorescent probe described herein was transferred into host cells; 2) Culture the host cells under conditions suitable for expression of the expression vector. 3) Separate spermidine fluorescent probes.

[0032] This invention also provides a method for detecting spermidine in a sample, comprising: contacting the sample with a fluorescent probe or fusion peptide described herein, or a fluorescent probe or fusion peptide prepared as described herein, and detecting changes in the optically active peptide. The detection can be performed in vivo, in vitro, subcellular, or in situ. The sample may be, for example, serum.

[0033] This article also provides a method for quantifying spermidine in a sample, comprising: contacting a sample with a fluorescent probe or fusion peptide described herein or a fluorescent probe or fusion peptide prepared by the method described herein, detecting changes in an optically active peptide, and quantifying spermidine in the sample based on the changes in the optically active peptide.

[0034] This invention also provides a method for screening compounds (e.g., drugs), comprising: contacting a candidate compound with a fluorescent probe or fusion peptide described herein, or a fluorescent probe or fusion peptide prepared as described herein, in a spermidine-containing system; detecting changes in the optically active peptide; and screening the compound based on the changes in the optically active peptide. This method can screen compounds in high throughput.

[0035] In one or more embodiments, the host cells described herein are contacted with the candidate compound in a spermidine-containing system, and the fluorescence change of the optically active peptide indicates whether the candidate compound can regulate the cell's uptake of spermidine.

[0036] Another aspect of the present invention provides a method for intracellular and / or extracellular localization of the spermidine, comprising: contacting a spermidine-containing system with the fluorescent probe or the host cell, and detecting fluorescence changes of the optically active peptide.

[0037] In one or more embodiments, the system is a solution system, a cellular system, or a subcellular system.

[0038] Another aspect of the present invention provides the application of the spermidine fluorescent probes, fusion peptides, or host cells described herein in the detection of spermidine in samples, screening of compounds, or intracellular / extracellular localization of spermidine. In one or more embodiments, the localization is real-time localization.

[0039] The beneficial effects of this invention are as follows: The spermidine fluorescent probe provided by this invention is easy to mature, exhibits large fluorescence dynamics and good specificity, and can be expressed in cells through gene manipulation. It enables real-time, high-throughput, and quantitative detection of spermidine both inside and outside cells, eliminating time-consuming sample processing steps. Experimental results show that the spermidine fluorescent probe provided in this application achieves a response to spermidine that is more than 10 times that of the control. Furthermore, it can perform localization, qualitative, and quantitative detection of spermidine in subcellular structures such as the cytoplasm, mitochondria, nucleus, Golgi apparatus, endoplasmic reticulum, and cell membrane. It also allows for high-throughput compound screening and quantitative detection of spermidine in blood. Attached Figure Description

[0040] Figure 1 This is an SDS-PAGE image of the exemplary spermidine fluorescent probe described in Example 2; Figure 2 The fluorescence spectrum of the exemplary spermidine fluorescent probe described in Example 3; Figure 3 This is a titration curve of the response of the exemplary spermidine fluorescent probe described in Example 3 to different concentrations of spermidine; Figure 4 This is a bar chart showing the specificity of the exemplary spermidine fluorescent probe described in Example 3 for the detection of 14 spermidine analogues; Figure 5 The kinetic curves of the exemplary spermidine fluorescent probe described in Example 3 in response to spermidine in the presence or absence of calcium or magnesium ions; Figure 6 The kinetic curves of the exemplary spermidine fluorescent probe described in Example 3 in response to spermidine at different pH values ​​are shown. Figure 7 This is a schematic diagram showing the fluorescence ratio of the exemplary spermidine fluorescent probe and the inactivated probe described in Example 3 at different pH values; Figure 8 This is a fluorescence imaging image of the subcellular organelle localization of the exemplary spermidine fluorescent probe described in Example 4 in mammalian cells; Figure 9 This is a schematic diagram illustrating the dynamic monitoring of spermidine concentration in the cytoplasm, nucleus, Golgi apparatus, and mitochondria of mammalian cells using an exemplary spermidine fluorescent probe, as described in Example 4. Figure 10 This is a schematic diagram illustrating the detection of changes in spermidine concentration caused by drug treatment in the cytoplasm, nucleus, Golgi apparatus, and mitochondria of mammalian cells using an exemplary spermidine fluorescent probe, as described in Example 5. Figure 11 The kinetic curves of the spermidine fluorescent probe described in Example 6 in response to spermidine in the cytoplasm, nucleus, Golgi apparatus and mitochondria of mammalian cells are shown. Figure 12 This is a schematic diagram illustrating the verification of the effect of the exemplary spermidine fluorescent probe on the action of polyamine transport inhibitors in mammalian cells, as described in Example 7. Figure 13 The bar chart shows a semi-quantitative analysis of polyamines in mouse blood using the exemplary spermidine fluorescent probe described in Example 8. Detailed Implementation

[0041] This invention provides a spermidine fluorescent probe, comprising a spermidine-sensitive polypeptide and an optically active polypeptide.

[0042] In this invention, the spermidine-sensitive polypeptide is any one of spermidine-binding protein, spermidine-binding protein functional variant 1, spermidine-binding protein functional variant 2, and spermidine-binding protein functional variant 3; the spermidine-sensitive polypeptide is derived from Pseudomonas aeruginosa; the amino acid sequence of the spermidine-binding protein is shown in SEQ ID NO:1; The spermidine-binding protein functional variant 1 is obtained by mutation of the spermidine-binding protein; the mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion. The mutation sites are any 1 to 5 of the following: T35, D36, P81, T134, V141, V147, P152, A157, E161, D180, E181, A185, N195, H197, K200, K203, F219, K223, Y239, D242, N269, W271, D273, P297, A301, Y309, L319, and Y331.

[0043] In this invention, the mutations are any combination of the following: F219 and N269; P81, F219 and N269; V141, F219 and N269; F219, K223 and N269; E181, F219, K223 and N269; T134, E161, F219 and N269; F219, N269, L319 and Y331; D180, F219, N269, L319 and Y331; T134, E161, D180, F219 and N269; T134, F219, N269 and L319; K200, F219, N269 and L319; E161, A185, F219 and N269; The mutations are as follows: P81 mutates to Q, T134 mutates to I, E161 mutates to I, D180 mutates to A, E181 mutates to A, A185 mutates to M, K200 mutates to I, F219 mutates to W or D, K223 mutates to Q, N269 mutates to G or P, L319 mutates to D or E, and Y331 mutates to G.

[0044] In this invention, the mutation is any combination of the following: F219D and N269G; F219W and N269G; P81Q, F219D and N269G; F219W, K223Q and N269G; E181A, F219W, K223Q and N269G; T134I, E161I, F219W and N269G; F219W, N269G, L319D and Y331G; D180A, F219W, N269G, L319D and Y331G; T134I, E161I, D180A, F219W and N269G; T134I, F219W, N269G and L319D; K200I, F219W, N269G and L319D; E161I, A185M, F219W and N269G; V141L, F219W and N269G.

[0045] In this invention, the spermidine-binding protein functional variant 2 is a truncated variant of amino acids 28 to 362 of the amino acid sequence of spermidine-binding protein functional variant 1. The spermidine-binding protein functional variant 3 is a nucleotide sequence that has ≥70% sequence similarity to spermidine-binding protein functional variant 1.

[0046] In this invention, the optically active polypeptide is located between residues 67-72, 218-224, or 337-342 of the spermidine-sensitive polypeptide; The optically active polypeptide is any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any one of the sequences containing 1 to 6 mutations at any of the Y1, N2, D4, K22, I27, F59, K62, S130, Y139, F199, and N205 sites in SEQ ID NO:2; The mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion.

[0047] In this invention, the mutation is any one or more of the following: Y1G, N2G, D4H, K22W, I27V, F59Y, K62A, S130R, Y139N, F199T and N205F; The optically active polypeptide is preferably located at one or more of the following sites of the spermidine-sensitive polypeptide: 67 / 68, 67 / 69, 67 / 70, 67 / 71, 67 / 72, 68 / 69, 68 / 70, 68 / 71, 68 / 72, 69 / 70, 69 / 71, 69 / 72, 70 / 71, 70 / 72, 71 / 72, 218 / 219, 218 / 220, 218 / 221, 218 / 222, 218 / 223, 218 / 224, 219 / 220, 219 / 221, 219 / 222, 219 / 223, 219 / 224, 220 / 221, 220 / 222, 220 / 223, 220 / 224, 221 / 222, 221 / 223, 22 1 / 224, 222 / 223, 222 / 224, 223 / 224, 337 / 338, 337 / 339, 337 / 340, 337 / 341, 337 / 342, 338 / 339, 338 / 340, 338 / 341, 338 / 342, 339 / 340, 339 / 341, 339 / 342, 340 / 341, 341 / 342; further preferably located at one or more of the following sites of the spermidine-sensitive polypeptide: 68 / 72, 69 / 70, 218 / 223, 219 / 221, 219 / 223, 219 / 224, 220 / 223, 220 / 224, 221 / 223, 221 / 224, 222 / 223, 339 / 340.

[0048] In this invention, the fluorescent probe comprises a spermidine-binding protein variant and an optically active polypeptide, and contains mutations selected from any of the following groups: (1) P81Q, F219D, and N269G of spermidine-binding protein; (2) F219W, K223Q, and N269G of spermidine-binding protein; (3) T134I, E161I, F219W, and N269G of spermidine-binding protein; (4) spermidine-binding protein variants. (5) Spermine-binding proteins F219W, N269G, L319D, and Y331G; (6) Spermine-binding proteins D180A, F219W, N269G, L319D, and Y331G; (7) Spermine-binding proteins T134I, E161I, D180A, F219W, and N269G. (8) Spermine-binding proteins T134I, F219W, N269G, and L319D; (9) Spermine-binding proteins K200I, F219W, N269G, and L319D; (10) Spermine-binding proteins E161I, A185M, F219W, and N269G; (11) Spermine-binding proteins V141L, F219W, and N269G; (12) Spermine-binding proteins The proteins P81Q, F219D and N269G, and the fluorescent proteins Y1G, N2G and D4H, (13) the spermidine-binding proteins T134I, E161I, F219W and N269G, and the fluorescent proteins Y1G, N2G and D4H, (14) the spermidine-binding proteins F219W, N269G, L319D and Y331G, and the fluorescent proteins Y1G, N2G and D4H.

[0049] In this invention, the amino acid sequence of the spermidine fluorescent probe is any one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0050] In this invention, the amino acid sequences of SEQ ID NO:1~18 are as follows: SEQ ID NO:1 (spuE): MQHSIGKTLLVAALATAIAGPVQAEKKSLHIYNWTDYIAPTTLKDFTKESGIDVSYDVFDSNETLEGKLVSGHSGYDIVVPSNNFLGKQIQAGAFQKLDKSKLPNWKNLDPALLKQLEVSDPGNQYAVPYLWGTNGIGYNVAKVKEVLGDQPIDSWAILFEPENMKKLAKCGVAFMDSGDEMLPAALNYLGDPNTHDPKDYKKAEEVLTKVRPYVSYFHSSKYISDLANGNICVAFGYSGDVFQAAARAEEAGKGIDIQYVIPKEGANLWFDLMAIPADAKAADNAYAFIDYLLRPEVIAKVSDYVGYANAIPGARPLMDKSVSDSEEVYPPQAVLDKLYVSAVLPAKVLRLQTRTWTRIKTGK; SEQ ID NO:2(cpVenus): YNSDNVYITADKQKNGICANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSFQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN; SEQ ID NO:3(cpGFP): NVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICCTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKIDFKEDGNILGHKLEYN; SEQ ID NO:4(cpmNeongreen): ADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYKGGSGGTVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTA; SEQ ID NO:5(cpYFP): YNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN; SEQ ID NO:6(cpBFP): NVYIKADKQKNGIKANFKIRHNIEGGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSILSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSESMVSKGEELFTGVVPIQVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLSHGVQCFSRYPDHMKQHDFFKSAMPGGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYN; SEQ ID NO:7(cpmCherry): VSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDTTSHNEDYTIVEQYERAEGRHSTGGMDELYKGGTGGSLVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEA; SEQ ID NO:8(cpmApple): VSERMYPEDGALKSEIKKGLRLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQCERAEGRHSTGGMDELYKGGTGGSMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPPDGPVMQKKTMGWEA; SEQ ID NO:9(cpLSSmScarlet2): LEADTERLYPEDGVLKGDIKMALRLKGGGRYLAHVRTTYKAKKPVLMPGAYNVDRKLDITSHNEDYTVVEQFERSEGRHSTGDMDELYKGGSGGTMVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYHKQSFPEGFKWERVMNFEDGGAVTVTQDTSLEDGTLIYEVKLRGTNFPPDGPVMQKKTMG; SEQ ID NO:10(spuE-220 / 223-cpVenus-T134I / E161I / F219W / N269G,GRISPD-H): ; SEQ ID NO:11(spuE-220 / 223-cpVenus-P81Q / F219D / N269G,GRISPD-C): ; SEQ ID NO:12(spuE-220 / 223-cpVenus-T134I / E161I / F219W / N269G / D273A,dGRISPD) 。

[0051] SEQ ID NO:13(CLS-GRISPD-C): ; SEQ ID NO:14(GRISPD-C-NLS): ; SEQ ID NO:15(Mito-GRISPD-C): MLSALVRPVSAALRRSFSTSAQNNGSGSLHIYNWTDYIAPTTLKDFTKESGIDVSYDVFDSNETLEGKLVSGHSGYDIVVQSNNFLGKQIQAGAFQKLDKSKLPNWKNLDPALLKQLEVSDPGNQYAVPYLWGTNGIGYNVAKVKEVLGDQPIDSWAILFEPENMKKLAKCGVAFMDSGDEMLPAALNYLGLDPNTHDPKDYKKAEEVLTKVRPYVSYDHGGSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSFQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNGGKYISDLANGNICVAFGYSGDVFQAAARAEEAGKGIDIQYVIPKEGANLWFDLMAIPADAKAADNAYAFIDYLLRPEVIAKVSDYVGYANAIPGARPLMDKSVSDSEEVYPPQAVLDKLYVSAVLPAKVLRLQTRTWTRIK; SEQ ID NO:16(Golgi-GRISPD-C): ; SEQ ID NO:17(ER-GRISPD-C): ; SEQ ID NO:18(MEM-GRISPC): .

[0052] The present invention also provides a nucleotide sequence encoding the spermidine fluorescent probe.

[0053] The present invention also provides the application of the spermidine fluorescent probe or the nucleotide sequence encoding the spermidine fluorescent probe in the localization or quantitative detection of spermidine.

[0054] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0055] The terms “comprising,” “including,” and their equivalents as used herein include the meanings of “containing” and “composed of,” for example, a composition “comprising” X may consist of only X or may contain other substances, such as X+Y.

[0056] As used herein, the term "spermidine-sensitive peptide" or "spermidine-responsive peptide" refers to a peptide that responds to spermidine, including any response to chemical, biological, electrical, or physiological parameters of the peptide in relation to the interaction with the sensitive peptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of the peptide, and changes in the primary, secondary, or tertiary structure of the peptide, including, for example, changes in protonation, electrochemical potential, and / or conformation. "Conformation" is the three-dimensional arrangement of the primary, secondary, and tertiary structures of a molecule containing side groups; a conformational change occurs when the three-dimensional structure of the molecule changes. Examples of conformational changes include a change from an α-helix to a β-sheet or vice versa. It is understood that a detectable change need not be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The spermidine-sensitive peptides described herein may also include their functional variants. Functional variants of spermidine-sensitive peptides include, but are not limited to, variants that can interact with spermidine to undergo the same or similar changes as the parent spermidine-sensitive peptide.

[0057] As used herein, the term "fluorescent probe" refers to a spermidine-sensitive peptide fused to an optically active peptide (e.g., a fluorescent protein), which is operatively inserted into the spermidine-sensitive peptide. The inventors discovered that when an optically active peptide is fused to a spermidine-sensitive peptide, such as a spermidine-binding protein, the conformational change resulting from the specific binding of the spermidine-sensitive peptide to physiological concentrations of spermidine induces a conformational change in the optically active peptide (e.g., a fluorescent protein), thereby altering the optical properties of the optically active peptide. By plotting standard curves using the fluorescence intensity of the fluorescent protein measured at different spermidine concentrations, the presence and / or concentration level of spermidine can be detected and analyzed.

[0058] The spermidine-sensitive polypeptides described in this invention include, but are not limited to, spermidine-binding protein spuE or variants thereof with more than 90% homology. The exemplary spermidine-binding protein spuE described in this invention is derived from *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa An exemplary spuE protein is shown in SEQ ID NO:1, and an exemplary truncated variant of the spuE protein is the fragment containing amino acids 28-362 of SEQ ID NO:1. When describing the fluorescent probes, spermidine-sensitive peptides, or spermidine-binding proteins of the present invention (e.g., when describing insertion sites or mutation sites), the amino acid residue numbers are referred to in SEQ ID NO:1.

[0059] A protein-based "optically active peptide" is a peptide with fluorescence emission capability. Fluorescence is an optical property of an optically active peptide, which can be used as a means of detecting the responsiveness of the fluorescent probe of the present invention. As used herein, the term "optical property" refers to the molar extinction coefficient at an appropriate excitation wavelength, fluorescence quantum yield, shape of the excitation or emission spectrum, maximum excitation wavelength and maximum emission wavelength, amplitude of excitation at two different wavelengths, ratio of emission amplitude at two different wavelengths, excited-state lifetime, or fluorescence anisotropy. A measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrate of the present invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative optical property, for example, fluorescence intensity at a specific wavelength or the integral of fluorescence intensity over the emission spectrum. Preferably, the protein substrate is selected to have optical properties that are easily distinguishable between inactive and activated conformational states. The optically active peptides described herein may also include their functional variants. Functional variants of optically active peptides include, but are not limited to, variants that can undergo changes in the same or similar optical properties as the parent optically active peptide.

[0060] In this article, "response fold" refers to the normalized fluorescence ratio. The greater the deviation of the probe's response fold from 1 (whether it increases or decreases), the greater the change in the probe's response to the substrate relative to the control, or the greater its responsiveness. For example, in this application, the normalized ratio is measured by detecting the change in the ratio of fluorescence intensity at 550 nm emission from 405 nm excitation to 550 nm emission from 488 nm excitation. 488 / 405 , R 488 / 405 The response fold was calculated as follows: Fluorescence signal values ​​were corrected by subtracting the detection signal values ​​from cells not expressing the probe protein. pH-sensitive interference was eliminated by dividing the probe detection signals from the parallel experimental groups by the inactivated probe detection signals from the control group to obtain corrected data.

[0061] F = F Sample -F Blank

[0062] F Corrected =F Sensor / F dSensor

[0063] R=R 488 / 405 =Ex 488 / Ex 405

[0064] R corrected = R sensor / R dSensor

[0065] Normalized R = R corrected (Spermidine) / R corrected (Buffer)

[0066] F represents fluorescence intensity. sample F represents the total fluorescence intensity of the sample expressing the fluorescent probe. Blank F represents the background fluorescence intensity of samples without expressed fluorescent probes. dSensor This represents the fluorescence intensity of the inactivating probe used as a pH control. Ex 488 Ex represents the fluorescence intensity of a fluorescent protein sample excited at 488 nm and emitted at 550 nm. 405 R represents the fluorescence intensity emitted at 550 nm when the fluorescent protein sample is excited at 405 nm. Sensor R represents the ratio of the fluorescence intensity of the probe. dSensor This represents the ratio of fluorescence intensity of the control inactivated probe. Normalized R 488 / 405 It is the multiple of the probe change or the multiple of the response. Normalized R 488 / 405 The greater the deviation from 1 (whether it increases or decreases), the greater the change factor or response factor of the probe.

[0067] "Connector" or "linking region" refers to an amino acid or nucleotide sequence that links two parts in a polypeptide, protein, or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the linking region between the spermidine-sensitive polypeptide and the optically active polypeptide is selected to be 0-6, and the number of amino acids at the carboxyl terminus is selected to be 0-6. When the recombinant fluorescent probe is used as a basic unit to link with a functional protein, it can be fused to the amino acid or carboxyl terminus of the recombinant fluorescent probe. The adapter sequence can be a short peptide chain composed of one or more flexible amino acids, such as glycine or serine.

[0068] The terms "chromophore," "fluorophore," and "fluorescent protein" used herein are synonymous, referring to proteins that emit fluorescence under excitation light. Fluorescent proteins are fundamental detection methods in the field of bioscience. Examples include the commonly used green fluorescent protein GFP and its cyclically rearranged derivatives such as blue fluorescent protein (cpBFP), green fluorescent protein (cpGFP), yellow-green fluorescent protein (cpVenus), and yellow fluorescent protein (cpYFP); as well as the commonly used red fluorescent protein RFP and its cyclically rearranged derivatives, such as cpmApple, cpmCherry, cpLSSmScarlet2, and cpmNeongreen derived from the bright monomeric green fluorescent protein mNeongreen. The sequences of exemplary fluorescent proteins are shown in any of SEQ ID NO:2-9. The spermidine fluorescent probe of this invention comprises a spermidine-sensitive polypeptide B, such as spermidine-binding protein or a variant thereof, and an optically active polypeptide A, such as a fluorescent protein. Optically active peptide A is inserted into spermidine-sensitive peptide B, dividing B into a first part B1 and a second part B2, forming a probe structure of type B1-A-B2; the interaction between spermidine-sensitive peptide B and spermidine leads to an increase in the fluorescence signal of optically active peptide A.

[0069] In the fluorescent probe of the present invention, the optically active polypeptide can be located at any position of the spermidine-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located in the following regions of the spermidine-sensitive polypeptide in the NC direction: amino acid residues 67-72, 218-224, and 337-342. For example, the optically active peptide is located at amino acid sequences 67 / 68, 67 / 69, 67 / 70, 67 / 71, 67 / 72, 68 / 69, 68 / 70, 68 / 71, 68 / 72, 69 / 70, 69 / 71, 69 / 72, 70 / 71, 70 / 72, 71 / 72, 218 / 219, 218 / 220, 218 / 221, 218 / 222, 218 / 223, 218 / 224, 219 / 220, 219 / 221, 219 / 222, 219 / 223, 219 / 223, 219 / 224 of the spermidine-binding protein. 9 / 224, 220 / 221, 220 / 222, 220 / 223, 220 / 224, 221 / 222, 221 / 223, 221 / 224, 222 / 223, 222 / 224, 223 / 224, 337 / 338, 337 / 339, 337 / 340, 337 / 341, 337 / 342, 338 / 339, 338 / 340, 338 / 341, 338 / 342, 339 / 340, 339 / 341, 339 / 342, 340 / 341, 341 / 342.

[0070] In this paper, at sites represented in the form of "X / Y", the two ends of the optically active polypeptide are respectively linked to partial spermidine-sensitive polypeptide fragments. The N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any amino acid from position 1 to position 28) to the X-terminal amino acid of the spermidine-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Y-terminal amino acid to the C-terminal ending amino acid (e.g., any amino acid from position Y to position 362) of the spermidine-sensitive polypeptide sequence. In this context, if the two numbers in the "X / Y" format are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by those numbers. For example, insertion site 67 / 68 indicates that the optically active polypeptide is located between amino acids 67 and 68 of the spermidine-sensitive polypeptide. If the two numbers in the "X / Y" format are not consecutive integers and X is less than Y, it indicates that the optically active polypeptide replaces the amino acids between those numbers. For example, insertion site 67 / 72 indicates that the optically active polypeptide replaces amino acids 68-71 of the spermidine-sensitive polypeptide. If X in the "X / Y" format is greater than or equal to Y, it indicates that the spermidine-sensitive polypeptide portion located at the N-terminus of the optically active polypeptide is located at the spermidine-sensitive end. The Xth amino acid of the polypeptide sequence terminates, while the spermidine-sensitive polypeptide portion located at the C-terminus of the optically active polypeptide begins at the Yth amino acid of the spermidine-sensitive polypeptide sequence; for example, insertion site 220 / 218 indicates that the N-terminus of the optically active polypeptide is fused with the N-terminal starting amino acid (e.g., any amino acid from position 1 to 28) to the 220th amino acid of the spermidine-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is fused with the 218th amino acid to the C-terminal ending amino acid (e.g., the 362nd amino acid) of the spermidine-sensitive polypeptide sequence, with an exemplary structure of: (amino acids 28 to 220 of the spermidine-sensitive polypeptide sequence) - (optically active polypeptide) - (amino acids 218 to 362 of the spermidine-sensitive polypeptide sequence).

[0071] In one or more embodiments, the fluorescent probe comprises, from the N-terminus to the C-terminus, residues 28-X of SEQ ID NO:1, an optically active polypeptide or a variant thereof shown in any one of SEQ ID NO:2-9, and residues Y-362 of SEQ ID NO:1, wherein X and Y are selected from any group of: (1) X is 67, Y is 68. (2) X is 67, Y is 69. (3) X is 67, Y is 70. (4) X is 67, Y is 71. (5) X is 67, Y is 72. (6) X is 68, Y is 69. (7) X is 68, Y is 70. (8) X is 68, Y is 71. (9) X is 68, Y is 72. (10) X is 69, Y is 70. (11) X is 69, Y is 71. (12) X is 69, Y is 72. (13) X is 70, Y is 71. (14) X is 70. Y is 72, (15) X is 71, Y is 72, (16) X is 218, Y is 219, (17) X is 218, Y is 220, (18) X is 218, Y is 221, (19) X is 218, Y is 222, (20) X is 218, Y is 223, (21) X is 218, Y is 224, (22) X is 219, Y is 220, (23) X is 219, Y is 221, (24) X is 219, Y is 222, (25) X is 219, Y is 223, (26) X is 219, Y is 224, (27) X is 220, Y is 221, (28) X is 220 Y is 222, (29) X is 220, Y is 223, (30) X is 220, Y is 224, (31) X is 221, Y is 222, (32) X is 221, Y is 223, (33) X is 221, Y is 224, (34) X is 222, Y is 223, (35) X is 222, Y is 224, (36) X is 223, Y is 224, (37) X is 337, Y is 338, (38) X is 337, Y is 339, (39) X is 337, Y is 340, (40) X is 337, Y is 341, (41) X is 337, Y is 342, (42) X is 338, Y is 339, (43) X is 338, Y is 340, (44) X is 338, Y is 341, (45) X is 338, Y is 342, (46) X is 339, Y is 340, (47) X is 339, Y is 341, (48) X is 339, Y is 342, (49) X is 340, Y is 341, (50) X is 340, Y is 342, (51) X is 341, Y is 342 Preferably, the optically active polypeptide is located at amino acid positions 68 / 72, 69 / 70, 218 / 223, 219 / 221, 219 / 223, 219 / 224, 220 / 223, 220 / 224, 221 / 223, 221 / 224, 222 / 223, and 339 / 340 of the spermidine-binding protein.

[0072] When referring to a polypeptide or protein, the term "variant" or "mutant" as used in this invention includes variants that have the same function as the polypeptide or protein but with a different sequence. Variants of polypeptides or proteins may include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more (typically 1-30, preferably 1-20, more preferably 1-10, most preferably 1-5) amino acids in the sequence of the polypeptide or protein, and sequences obtained by adding one or more (typically up to 20, preferably up to 10, more preferably up to 5) amino acids to its carboxyl terminus and / or amino terminus. In some mutants, amino acid residues are altered without changing the overall conformation and function of the polypeptide or protein, i.e., functionally conserved mutations. For example, in the art, substitution with amino acids of similar or comparable properties generally does not change the function of the polypeptide or protein. In the art, amino acids of similar properties often refer to amino acid families with similar side chains, which are well-defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, histidine). For example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not alter the function of the polypeptide or protein. For many common, known, non-genetically encoded amino acids, conserved amino acid substitutions are known in the art; for other non-coding amino acids, conserved substitutions can be determined based on a comparison of their physicochemical properties with those of genetically encoded amino acids. As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues to the terminus of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6His or Flag, or proteolytic sites of factor Xa, thrombin, or enterokinase. Polypeptide or protein variants can include: homologous sequences, conserved variants, allelic variants, natural mutants, and induced mutants.These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the stated polypeptide or protein. An exemplary truncated variant of the spuE protein is the fragment of SEQ ID NO:1 containing amino acids 28-362, which preserves the binding function of the spuE protein to spermidine and does not affect the optical property changes of the inserted optically active polypeptide in response to spermidine binding.

[0073] The fluorescent probes of this invention may comprise mutated spermidine-sensitive peptides. Spermine-binding protein variants with mutations at sites selected from the following sites in SEQ ID NO:1 or a truncated variant thereof exhibit different binding activity than spermidine: T36, D36, P81, D180, E181, F219, K223, N269, W271, D273, and Y309. The amino acid mutations include modifications, substitutions, or deletions of amino acids. In a preferred embodiment, the mutations of the spermidine-binding protein variants include mutations at sites selected from any of the following groups: (1) P81 and N269, (2) D180 and N269, (3) E181 and N269, (4) F219 and N269, (5) K223 and N269, (6) N269 and Y309, (7) N269 and W271, (8) N269 and D273, (9) D180, F219 and N269, (11) E181, F219 and N269, (12) P81, F219, K223 and N269.

[0074] In one or more embodiments, as an example, in SEQ ID NO:1 or a truncated variant thereof, T35 mutates to G. In one or more embodiments, D36 mutates to G. In one or more embodiments, P81 mutates to Q. In one or more embodiments, D180 mutates to A. In one or more embodiments, E181 mutates to A. In one or more embodiments, F219 mutates to D or W. In one or more embodiments, K223 mutates to Q. In one or more embodiments, N269 mutates to G or P. In one or more embodiments, W271 mutates to R or A. In one or more embodiments, D273 mutates to A. In one or more embodiments, Y309 mutates to A.

[0075] In one or more embodiments, the mutation comprises mutations selected from any of the following groups: (1) F219D and N269G, (2) F219W and N269G, (3) F219W, K223Q and N269G, (4) P81Q, F219D and N269G, (5) D180A, F219W and N269G, (6) and E181A, F219W, N269G, (7) F219D and N269P, (8) N269G and W271Q, (9) N269G and D273A, (10) N269G and W271R. The present invention provides spermine-binding protein variants having these mutations and fluorescent probes comprising such spermine-binding protein variants as spermine-sensitive peptides.

[0076] The fluorescent probe of the present invention may comprise a mutated optically active polypeptide. In some embodiments, the mutated optically active polypeptide has the sequence shown in SEQ ID NO:2 and has mutations at one, two, three, four, five, or six of the following sites: Y1, N2, D4, K22, I27, S130, Y139, F199, N205, wherein the mutations include amino acid modifications, substitutions, or deletions. In one or more embodiments, the mutations include mutations selected from any of the following sites: Y1, N2, D4, K22, I27, S130, Y139, F199, N205. Specifically, the mutations are Y1G, N2G, D4H, K22W, I27V, F59Y, K62A, S130R, Y139N, F199T, and N205F; In one or more embodiments, the fluorescent probe comprises any of the amino acid sequences SEQ ID NO:10-17 or variations thereof. In one or more embodiments, the fluorescent probe provided by the present invention comprises a sequence having 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity with any of the amino acid sequences SEQ ID NO:10-31. In a preferred embodiment, the fluorescent probe provided by the present invention comprises a sequence substantially similar to or identical to any of the amino acid sequences SEQ ID NO:10-31. Preferably, the fluorescent probe has the sequence shown in SEQ ID NO:10-18; more preferably, the fluorescent probe has the sequence shown in SEQ ID NO:10-18.

[0077] In some specific embodiments, the spermidine-sensitive peptide in the fluorescent probe is shown as amino acids 28-362 of SEQ ID NO:1, and the optically active peptide is shown as SEQ ID NO:2, the optically active peptide being located at position 220 / 223 of the spermidine-sensitive peptide, and the spermidine-sensitive peptide having a mutation selected from any of the following: (1) P81Q, F219D, and N269G of spermidine-binding protein, (2) F219W, K223Q, and N269G of spermidine-binding protein, (3) T134I, E161I, F219W, and N2 of spermidine-binding protein. 69G, (4) spermidine-binding proteins F219W, N269G, L319D and Y331G, (5) spermidine-binding proteins E181A, F219W, K223Q and N269G, (6) spermidine-binding proteins D180A, F219W, N269G, L319D and Y331G, (7) spermidine-binding proteins T134I, E161I, D180A, F219W (8) Spermine-binding proteins T134I, F219W, N269G and L319D, (9) Spermine-binding proteins K200I, F219W, N269G and L319D, (10) Spermine-binding proteins E161I, A185M, F219W and N269G, (11) Spermine-binding proteins V141L, F219W and N269G, (12) Spermine Amine-binding proteins P81Q, F219D and N269G, and fluorescent proteins Y1G, N2G and D4H, (13) spermidine-binding proteins T134I, E161I, F219W and N269G, and fluorescent proteins Y1G, N2G and D4H, (14) spermidine-binding proteins F219W, N269G, L319D and Y331G, and fluorescent proteins Y1G, N2G and D4H.

[0078] In two or more polypeptide or nucleic acid molecular sequences, the term "identity" or "percentage of identity" refers to the fact that, when compared and matched for maximum correspondence using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection, two or more sequences or subsequences are identical or have a certain percentage of amino acid residues or nucleotides identical in a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical). For example, the preferred algorithms for determining the percentage of sequence identity and the percentage of sequence similarity are BLAST and BLAST 2.0 algorithms, which can be found in Altschul et al. (1977) Nucleic Acids Res .25:3389 and Altschul et al. (1990) J .Mol.Biol .215:403, respectively.

[0079] As is known to those skilled in the art, gene cloning often requires the design of suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, without affecting the activity of the target polypeptide or protein. Similarly, to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside host cells, or facilitate the purification of recombinant proteins, it is often necessary to add certain amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein. These include, but are not limited to, suitable adaptor peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E-binding proteins, protein A, tags such as 6×His or Flag, or proteolytic enzyme sites such as factor Xa, thrombin, or enterokinase.

[0080] As used herein, the terms "functional variant," "derivative," and "analyte" refer to a protein that substantially retains the same biological function or activity as the original polypeptide or protein (e.g., spuE protein or fluorescent protein). The functional variants, derivatives, or analogs of the polypeptides or proteins (e.g., spuE protein or fluorescent protein) of this invention can be...

[0081] (i) A protein in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or

[0082] (ii) Proteins having substituent groups in one or more amino acid residues, or

[0083] (iii) A protein formed by fusing a mature protein with another compound (such as a compound that extends the protein's half-life, like polyethylene glycol), or

[0084] (iv) Proteins formed by fusing additional amino acid sequences to this protein sequence (such as secretory sequences or sequences used to purify this protein or proteogen sequences, or fusion proteins formed with fragments of antigen IgG). These functional variants, derivatives, and analogs are well known to those skilled in the art in accordance with the teachings herein.

[0085] The difference between the analogue and the original polypeptide or protein can be a difference in amino acid sequence, a difference in modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis through radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.

[0086] The analogues also include those having residues different from naturally occurring L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the spermidine-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogues listed above. Modifications (generally without altering the primary structure) include chemically derived forms of proteins, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during protein synthesis and processing or further processing steps. This modification can be accomplished by exposing the protein to glycosylation enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility are also included.

[0087] The fusion peptide of this invention comprises the fluorescent probe described herein and other peptides. In some embodiments, the fluorescent probe described herein further comprises other peptides fused thereto. These other peptides do not affect the properties of the fluorescent probe. The other peptides may be located at the N-terminus and / or C-terminus of the fluorescent probe. In some embodiments, the other peptides include peptides for targeting the fluorescent probe to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. A linker may be present between the fluorescent probe and other peptides in the fusion peptide described herein.

[0088] The subcellular organelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, tags used for purification or for immunoblotting include 6-histidine (6×His), glutathione S-transferase (GST), and flag-tags.

[0089] This invention also provides a method for preparing the above-mentioned spermidine fluorescent probe, comprising the following steps: 1) The nucleic acid sequence encoding the spermidine fluorescent probe described in this paper is incorporated into the expression vector; 2) Transfer the expression vector into host cells; 3) Culture the host cells under conditions suitable for expression of the expression vector. 4). Separate spermidine fluorescent probes.

[0090] As used herein, the term "nucleic acid" or "nucleotide" can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" as used herein can be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is a substitution of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes. The nucleic acids of this invention may comprise a nucleotide sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity with the described nucleic acid sequence. This invention also relates to nucleic acid fragments that hybridize with the sequences described above. As used herein, a “nucleic acid fragment” contains at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).

[0091] The full-length sequence or fragments of the fluorescent probe or fusion protein of this invention can generally be obtained by PCR amplification, artificial synthesis, or recombinant methods. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed in this invention, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequence. When the nucleotide sequence is greater than 2500 bp, it is preferable to perform 2 to 6 PCR amplifications, and then splice the fragments from each amplification in the correct order. This invention does not impose any special limitations on the PCR amplification procedure and system; conventional PCR amplification procedures and systems in the art can be used. Recombinant methods can also be used to obtain the relevant sequence in large quantities. This typically involves cloning it into a vector, transforming it into cells, and then isolating and purifying the relevant polypeptide or protein from the proliferated host cells using conventional methods. Furthermore, the relevant sequence can also be synthesized artificially, especially when the fragment length is short. In this invention, when the nucleotide sequence of the fluorescent probe is less than 2500 bp, artificial synthesis can be used. The artificial synthesis method is a conventional DNA synthesis method in the art, without any other special requirements. Typically, long sequences are obtained by first synthesizing multiple small fragments and then ligating them. Currently, the DNA sequence encoding the protein of this invention (or its functional variants, derivatives, or analogues) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of this invention using methods such as mutagenic PCR or chemical synthesis.

[0092] After obtaining the nucleotide sequence encoding a fluorescent probe, this invention incorporates the nucleotide sequence encoding the fluorescent probe into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" used herein are used interchangeably and refer to prokaryotic or eukaryotic vectors well-known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors. These vectors can replicate and stably express in a host cell. An important characteristic of these recombinant vectors is that they typically contain an expression control sequence. The term "expression control sequence" used herein refers to an element that can be operatively linked to the target gene to regulate the transcription, translation, and expression of the target gene. This can be an origin of replication, promoter, marker gene, or translation control element, including enhancers, operons, terminators, ribosome binding sites, etc. The choice of expression control sequence depends on the host cell used. Recombinant vectors applicable in this invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operative linking" refers to the connection of the target nucleotide sequence to a regulatory sequence in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods for constructing expression vectors containing the coding sequence of the fusion protein of this invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or trp promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV promoter, the HSV thymidine kinase promoter, the SV40 early and late promoters, the retroviral LTR, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one or more embodiments, the expression vector can be a commercially available pET28a+ vector, with no other special requirements. Exemplarily, the nucleotide sequence encoding the fluorescent probe and the expression vector are double-digested with BamHI and XhoI, respectively, and then the digestion products are ligated to obtain the recombinant expression vector. This invention does not specifically limit the specific steps and parameters of digestion and ligation; conventional steps and parameters in the art can be used.

[0093] After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or polypeptide including a fusion protein. This transfer process can be performed using conventional techniques well known to those skilled in the art, such as transformation or transfection. The host cell described in this invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, serving as the site for recombinant gene amplification. Ideally, the recipient cell should meet the conditions of easy acquisition and proliferation. The "host cell" of this invention can include prokaryotic and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. Specifically, it can be bacterial cells of *Escherichia coli*, *Streptomyces*, and *Salmonella typhimurium*, fungal cells such as yeast, plant cells, insect cells of *Drosophila S2* or *Sf9*, animal cells such as CHO, HeLa, U2-OS, U937, THP-1, and 293T, etc., including but not limited to the aforementioned host cells. The host cell is preferably a cell type well-suited for gene product expression or fermentation production, such cells are well known and commonly used in the art. An exemplary host cell used in the embodiments of this invention is *Escherichia coli* strain BL21-DE3. Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0094] The methods for transferring DNA to host cells described in this invention are conventional methods in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-glucan-mediated transfection, liposome transfection, chemocompetent cells, chemically mediated transfer, or electroporation. When the host is a prokaryote such as *Escherichia coli*, the preferred method is the CaCl2 or MnCl2 method, and the steps used are well-known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be used: PEI method, electroporation, liposome transfection, etc.

[0095] This invention involves transforming an expression vector into host cells, followed by amplification and expression culture of the host cells to isolate the spermidine fluorescent probe. The host cell amplification and expression culture can be performed using conventional methods. Depending on the type of host cells used, the culture medium can be any conventional medium. Culture is carried out under conditions suitable for host cell growth.

[0096] In this invention, the fluorescent probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated or purified using various separation methods based on its physical, chemical, and other properties. This invention does not specifically limit the method for separating the spermidine fluorescent protein; conventional methods for separating fusion proteins in the art can be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional refolding treatment, salting out, centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography, affinity chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof. In one or more embodiments, the fluorescent probe is separated using His-tagged affinity chromatography.

[0097] This invention also provides the application of the spermidine fluorescent probe in real-time localization, quantitative detection, and high-throughput compound screening of spermidine. In one aspect, the spermidine fluorescent probe is preferably linked to a signal peptide targeting different subcellular localizations, transferred into cells, and its real-time localization is achieved by detecting the intensity of the fluorescence signal in the cells; quantitative detection of spermidine is performed using a spermidine standard titration curve. The spermidine standard titration curve described in this invention is plotted based on the fluorescence signals of the spermidine fluorescent probe at different concentrations of spermidine. The spermidine fluorescent probe of this invention is directly transferred into cells, eliminating the need for time-consuming sample processing during real-time localization and quantitative detection of spermidine, thus improving accuracy. In high-throughput compound screening, the spermidine fluorescent probe of this invention adds different compounds to the cell culture medium and measures the changes in spermidine content to screen for compounds that affect changes in spermidine content. The applications of the spermidine fluorescent probe in real-time localization, quantitative detection, and high-throughput compound screening described in this invention are not for diagnostic or therapeutic purposes and do not involve the diagnosis or treatment of diseases.

[0098] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0099] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0100] 1. Experimental materials and reagents

[0101] The embodiments mainly employ conventional genetic engineering molecular biology cloning methods, cell culture, and imaging methods, which are well known to those skilled in the art. Examples include: Jane Rothcams et al., *Molecular Cloning: A Laboratory Manual* (3rd edition, August 2002, Science Press, Beijing), J. Sambrook and DW Russell, translated by Huang Peitang et al.; Fereschney et al., *Animal Cell Culture: A Basic Technical Guide* (5th edition), translated by Zhang Jingbo and Xu Cunshuan et al.; and J.S. Bonnie Fesnon, M. Dassault et al., *A Concise Laboratory Manual of Cell Biology*, translated by Zhang Jingbo et al.

[0102] The plasmids used in the examples, including pET28a(+)-spuE (SEQ ID NO:1, spuE), pET28a(+)-cpVenus (SEQ ID NO:2, cpVenus), pET28a(+)-cpGFP (SEQ ID NO:3, cpGFP), pET28a(+)-cpmNeongreen (SEQ ID NO:4, cpmNeongreen), pET28a(+)-cpYFP (SEQ ID NO:5, cpYFP), pET28a(+)-cpBFP (SEQ ID NO:6, cpBFP), pET28a(+)-cpmCherry (SEQ ID NO:7, cpmCherry), pET28a(+)-cpmApple (SEQ ID NO:8, cpmApple), and pET28a(+)-cpLSSmScarlet2 (SEQ ID NO:9, cpLSSmScarlet2), were constructed by the Li Xinjian research group at the Institute of Biophysics, Chinese Academy of Sciences. pET28a(+) and pLenti-CMV plasmids were purchased from Sigma-Aldrich. All primers used for PCR were synthesized, purified, and identified by mass spectrometry by Suzhou Genewise Biotechnology Co., Ltd. The expression plasmids constructed in the examples were sequenced by Suzhou Genewise Biotechnology Co., Ltd. Taq DNA polymerase used in each example was purchased from Beijing Polymer Biotechnology Co., Ltd., pfu DNA polymerase from Beijing TransGen Biotech Co., Ltd., and KOD DNA polymerase from TOYOBO Corporation, Japan. Both polymerases were purchased with corresponding polymerase buffers and dNTPs. Restriction endonucleases such as BamHI, HindIII, NdeI, XhoI, and EcoRI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from NEB Corporation, with corresponding buffers provided. Lipofectamine 3000 transfection reagent was purchased from Invitrogen. Spermine (#S1501), putrescine (#P6024), and spermine (#S4264) were all purchased from Sigma-Aldrich (USA). Unless otherwise stated, inorganic salts and other chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Ampicillin (Amp), kanamycin sulfate (Kana), and puromycin (Puro) were purchased from Aladdin Biochemical Technology Co., Ltd. 96-well and 384-well fluorescence detection plates were purchased from PerkinElmer. DMEM medium, fetal bovine serum, PBS, trypsin, penicillin, and streptomycin antibiotics required for cell culture were all purchased from Gibco (USA).

[0103] The DNA purification kit used in these examples was purchased from OMEGA, and the general plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. The ClonExpress homologous recombination kit was also used. The II One Step Cloning Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. Cloned strains DH5α, BL21, and Stbl3 were purchased from Beijing TransGen Biotech Co., Ltd. Nickel affinity chromatography column packing material was from Beijing Lamborghini Trading Co., Ltd.

[0104] The main instruments used in the examples include: Biotek Synergy H1 multi-functional microplate reader (BioTek, USA), AVANTI JXN-30 high-speed refrigerated centrifuge (Beckman, USA), Centrifuge 5424R refrigerated centrifuge (Eppendorf, Germany), Bio-RADT100 PCR amplification system (Thermo Fisher Scientific, USA), JN02c high-pressure homogenizer (Juning, China), LF-600 nucleic acid electrophoresis system (Beijing Longfang, China), NanoPhotometer NP80 fluorescence spectrophotometer (Implen, Germany), HERACELL 150i CO2 constant temperature cell culture incubator (Thermo Fisher Scientific, USA), IX73 inverted fluorescence microscope (OLYMPUS, Japan), BD LSRFortwssa flow cytometer (BD, USA), and Nikon A1R+ laser confocal microscope (Nikon, Japan).

[0105] II. Molecular Biology Methods and Cellular Experimental Methods

[0106] II.1 Polymerase Chain Reaction (PCR): 1. Target Fragment Amplification PCR: This method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system is as follows: template sequence 3 μL (400±100 ng), forward primer (20 μM) 1 μL, reverse primer (20 μM) 1 μL, 5×pfu buffer 10 μL, pfu DNA polymerase 1 μL, dNTP (10 mM) 4 μL, sterile ultrapure water (ddH2O) 30 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 95℃ for 2 minutes, 30 cycles (98℃ for 30 seconds, 55℃ for 30 seconds, 72℃ for a certain time (1000-2000 bp / min)), extension at 72℃ for 5 minutes.

[0107] 2. Long Fragment (>2500bp) Amplification PCR: The long fragment amplification used in this invention is mainly a reverse PCR amplification vector, a technique used in the following examples to obtain site-directed mutagenesis. Reverse PCR primers are designed at the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product then contains the corresponding mutation site. The long fragment amplification PCR reaction system is as follows: template sequence 1 μL (40±10 ng), forward primer (20 μM) 1 μL, reverse primer (20 μM) 1 μL, 10×KOD PCR buffer 5 μL, KOD DNA polymerase 1 μL, dNTP (2 mM) 5 μL, MgSO4 (25 mM) 3 μL, sterile ultrapure water (ddHO) 33 μL, total volume 50 μL. The PCR amplification program is as follows: denaturation at 94℃ for 2 minutes, 35 cycles (98℃ for 10 seconds, 55℃ for 30 seconds, 68℃ for a certain time (2000bp / min)), extension at 68℃ for 5 minutes; or denaturation at 94℃ for 2 minutes, 35 cycles (98℃ for 10 seconds, 68℃ for a certain time (2000bp / min)), extension at 68℃ for 10 minutes.

[0108] II.2 Nucleotide restriction enzyme digestion reaction: The double digestion system for the plasmid vector is as follows: 20 μL plasmid vector (approximately 8 μg), 5 μL 10× buffer, 1.5 μL restriction endonuclease A, 1.5 μL restriction endonuclease B, and sterile ultrapure water to a total volume of 50 μL. Reaction conditions: 37℃, 6 hours.

[0109] II.3 Ligation of Target Fragment and Vector Response Different fragments and vectors require different ligation methods. This invention primarily uses two ligation methods.

[0110] 1. PCR amplification was performed to ensure that the 5' and 3' ends of the insert fragment contained sequences (15-20 bp) identical to those at the ends of the linearized vector. The fragment was then recombinated into the vector using a homologous recombination kit. The homologous recombination system was as follows: 3.5 μL (75 ± 15 ng) of DNA fragment containing homologous arms, 3.5 μL (250 ± 50 ng) of linearized vector fragment, 2 μL of 5× homologous recombination buffer, 1 μL of homologous recombination enzyme, and sterile ultrapure water to a total volume of 10 μL. The reaction was carried out at 37°C for 30 minutes.

[0111] 2. Ligation of DNA fragments with sticky ends and vector fragments with sticky ends: DNA fragments digested by restriction endonucleases typically produce prominent sticky ends, which can then be ligated with vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system is as follows: 3.5 μL (75±15 ng) of the digested PCR product DNA fragment, 3.5 μL (250±50 ng) of the digested plasmid, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, and sterile ultrapure water to a total volume of 10 μL. Reaction conditions: 16℃, 12 hours.

[0112] II.4 Preparation and Transformation of Competent Cells: 1. Inoculate the bacteria into culture medium (1L dispensed into 4 bottles, the volume of the culture should not exceed one-quarter of the volume, 250ml in a 1L Erlenmeyer flask) and incubate overnight at 18°C ​​or room temperature at 200rpm. 2. Measure OD600 in the morning. If it reaches 0.45, incubate the culture medium on ice for 10 minutes. 3-4℃, 2500g for 10 minutes; 4. Resuspend each bottle (1L) in 320ml of cold Inoue solution; 5. 4℃, 2500g for 10 minutes; 6. Resuspend each bottle (1L) in 80ml of cold Inoue solution; 7. Add DMSO to a final concentration of 7% (add 1.5 mL for every 20 mL); 8. Incubate on ice for 10 minutes; 9. Dispense and quick-freeze with liquid nitrogen.

[0113] SOB medium: 5g NaCl, 20g tryptone, 5g yeast extract, 2.5ml 1M KCl (0.186g).

[0114] Inoue buffer: Add 10.9g MnCl2 (55mM), 2.2g CaCl2 (20mM), 18.7 KCl (250mM), and 20ml 0.5M PIPESbuffer (10mM) to water to 1L and filter.

[0115] 0.5M PIPES buffer: Dissolve 15.1g of PIPES in 80ml of water, add KOH until clear, adjust the pH to 6.7 with KOH and HCl, bring the volume to 100ml, dispense and freeze at -20℃.

[0116] Transformation of competent cells: 1. Take 100 μL of competent cells and thaw them on an ice bath.

[0117] 2. Add an appropriate volume of ligation product, gently mix by pipetting, and incubate on ice for 30 minutes. The volume of ligation product added is usually less than 1 / 10 of the competent cell volume.

[0118] 3. Place the bacterial solution in a 42℃ water bath for 90 seconds for heat shock, then quickly transfer it to an ice bath and place it for 5 minutes.

[0119] 4. Add 500 μL LB and incubate at 200 rpm for 1 hour on a constant temperature shaker at 37℃.

[0120] 5. Centrifuge the bacterial culture at 4000 rpm for 3 minutes, and keep 200 μL of supernatant. Spread the bacterial cells evenly on the surface of an agar plate containing appropriate antibiotics. Incubate the plate upside down in a 37°C incubator overnight.

[0121] II.5 Protein Expression, Purification, and Fluorescence Detection

[0122] 1. Transform the expression vector (e.g., a spermidine fluorescent probe expression vector based on pET28a(+)) into BL21(DE3) cells, incubate upside down overnight, and pick clones from the plate into shake tubes for overnight incubation. Inoculate the turbid bacterial culture into 1L of medium, place in a shaker at 37°C, and incubate at 220rpm until OD≈0.6~0.8. Add 1 / 1000 (v / v) IPTG (1M) and induce expression at 18°C ​​for 20-24 hours.

[0123] 2. After induction of expression, centrifuge at 4000 rpm for 10 minutes to collect the bacteria. Resuspend the bacterial pellet in protein purification buffer (25 mM HEPES pH 7.4, 400 mM NaCl, 10% glycerol, 5 mM β-ME, 1 mM PMSF) and autoclave until the bacterial cells are clear. Centrifuge at 16000 rpm at 4°C for 35 minutes.

[0124] 3. The supernatant from centrifugation was purified into protein using a self-assembled nickel affinity chromatography column. The protein after nickel affinity chromatography was desalted and further purified by size exclusion chromatography, and identified by SDS-PAGE. After SDS-PAGE identification, the purified protein was diluted with assay buffer (100 mM HEPES, 150 mM NaCl, pH 7.4) to a final concentration of 0.2–5 μM. Spermine was prepared into a stock solution with a final concentration of 50 mM using assay buffer (100 mM HEPES, 150 mM NaCl, pH 7.4).

[0125] 4. Take 100 μl of 1 μM protein solution, incubate at 37℃ for 10 minutes, add spermidine for titration, and measure the fluorescence intensity of the protein emitted at 550 nm after excitation by 405 nm light and at 550 nm after excitation by 488 nm light. The fluorescence excitation and emission measurements of the samples were performed using a multifunctional fluorescent microplate reader.

[0126] II.6 Transfection and fluorescence detection of mammalian cells: 1. The pLenti-CMV-based spermidine fluorescent probe plasmid was transfected into 293T cells using PEI transfection reagent and cultured in a cell culture incubator at 37°C and 5% CO2. Fluorescence detection was performed 48 hours after the exogenous gene was fully expressed.

[0127] 2. After the cells were found to fully express the probe, the adherent 293T cells were washed three times with PBS and placed in HBSS solution for detection by fluorescence microscopy and ELISA reader.

[0128] Example 1: Spermine-binding protein particles

[0129] The spuE(28-362) gene was amplified by PCR. After gel electrophoresis, the PCR product was recovered and digested with BamHI and XhoI enzymes. Simultaneously, the pET28a(+) vector was double-digested with the corresponding enzymes. Ligation was performed using T4 DNA ligase, and the product was used to transform DH5α cells. The transformed DH5α cells were plated on LB agar plates (100 μg / mL kanamycin) and incubated overnight at 37°C. Plasmids were extracted from the grown DH5α transformants and identified by PCR. Positive plasmids, after successful sequencing, were used for subsequent plasmid construction.

[0130] Example 2: Expression and detection of fluorescent probes at different cpVenus insertion sites

[0131] In this embodiment, the following sites were selected for insertion into cpVenus based on pET28a-spuE to obtain the corresponding pET28a-spuE-cpVenus plasmids: 67 / 68, 67 / 69, 67 / 70, 67 / 71, 67 / 72, 68 / 69, 68 / 70, 68 / 71, 68 / 72, 69 / 70, 69 / 71, 69 / 72, 70 / 71, 70 / 72, 71 / 72, 218 / 219, 218 / 220, 218 / 221, 218 / 222, 218 / 223, 218 / 224, 219 / 220, 219 / 221. , 219 / 222, 219 / 223, 219 / 224, 220 / 221, 220 / 222, 220 / 223, 220 / 224, 221 / 222, 221 / 223, 221 / 224, 222 / 223, 222 / 224, 223 / 224, 337 / 338, 337 / 339, 337 / 340, 337 / 341, 337 / 342, 338 / 339, 338 / 340, 338 / 341, 338 / 342, 339 / 340, 339 / 341, 339 / 342, 340 / 341, 341 / 342.

[0132] The DNA fragment of *cpVenus* and the linearized vector pET28a-spuE were amplified using PCR. The 5' and 3' ends of pET28a-spuE contained sequences (15-20 bp) completely identical to those at the ends of *cpVenus*. Homologous recombination of the linearized pET28a-spuE and the *cpVenus* fragment was performed using ClonExpress II. The product was transformed into DH5α cells, which were then plated on LB agar plates (100 μg / mL kanamycin) and incubated overnight at 37°C. Positive clones identified by PCR were subjected to plasmid extraction and sequencing. Sequencing was performed by Genewiz.

[0133] After successful sequencing, the recombinant plasmid was transformed into BL21(DE3) to induce expression, and the protein was purified. SDS-PAGE electrophoresis showed a size around 69 kDa. This size is consistent with the size of the pET28a-spuE-cpVenus fusion protein containing the His-tag purified label. Coomassie Brilliant Blue staining results are as follows. Figure 1 As shown.

[0134] Spermine response screening was performed using the supernatant of fragmented *E. coli* expressing the *spuE-cpVenus* fusion protein. The detection signal of the fusion protein containing spermine was divided by the fluorescence intensity detection signal of the fusion protein without spermine. The results are shown in Table 1. The detection results show that: The site with the strongest response to spermidine is 220 / 223, which is 4.09 times higher.

[0135] Table 1. Variable fold at different insertion sites

[0136] Example 3: Expression and detection of mutant fluorescent probes

[0137] A fluorescent probe mutant was constructed based on spuE-220 / 223-cpVenus. Mutations were performed on specific sites of plasmid pET-28a-spuE-220 / 223-cpVenus using circular PCR. Primers were randomly mutated at the desired mutation sites using the degenerate base NNK. The PCR products were excised using DpnI nuclease to obtain mutant plasmids at the T35, D36, P81, T134, V141, V147, P152, A157, E161, D180, E181, A185, N195, H197, K200, K203, F219, K223, Y239, D242, N269, W271, D273, P297, A301, Y309, L319, and Y331 sites. These mutant plasmids were transformed to construct libraries and tested. Mutants with a greater than 6-fold response to spermidine were screened, and sequencing was performed by Genewiz. The sequences of partially mutated fluorescent probes are shown in the table below. Exemplary nucleic acid sequences are shown in SEQ ID NO:10 (spuE-220 / 223-cpVenus-T134I / E161I / F219W / N269G) and SEQ ID NO:11 (spuE-220 / 223-cpVenus-P81Q / F219D / N269G).

[0138] Table 2. Sequences of the mutated fluorescent probes

[0139] Example 3: Characteristics of the fluorescent probe

[0140] The two spermidine fluorescent probes numbered 1 and 5 from Example 2 were treated with 0 mM and 10 mM spermidine for 10 minutes, respectively, and then their fluorescence spectra were detected using an ELISA reader.

[0141] Excitation spectrum determination: Excitation spectra were recorded with an excitation range of 360 nm to 520 nm and an emission wavelength of 550 nm, read every 2 nm. The results showed that the probe had two excitation peaks at approximately 420 and 500 nm.

[0142] Emission spectrum determination: With a fixed excitation wavelength of 488 nm, the emission spectrum from 500 to 550 nm was recorded, with readings taken every 2 nm. The excitation and emission spectra are as follows: Figure 2 As shown.

[0143] Kinetic curve determination: For the two spermidine fluorescent probes numbered 1 and 5 obtained in Example 2, spermidine concentration gradient (0~100mM) detection was performed to detect the change in the ratio of fluorescence intensity at 405nm excitation and 550nm emission to fluorescence intensity at 488nm excitation and 550nm emission. The Kd (binding constant) of these two spermidine fluorescent probes are 2.17 μM and 427.2 μM, respectively. Probe number 1 has high affinity and is suitable for detecting spermidine concentration in biological samples, named GRISPD-H (SEQ ID NO:10, spuE-220 / 223-cpVenus-T134I / E161I / F219W / N269G). Probe number 5 has moderate affinity and is suitable for detecting spermidine concentration in living cells, named GRISPD-C (SEQ ID NO:11, spuE-220 / 223-cpVenus-P81Q / F219D / N269G). The inactivated probe dGRISPD (SEQ ID NO:10) is obtained by mutating the probe with D273A. NO:12, spuE-220 / 223-cpVenus-T134I / E161I / F219W / N269G / D273A) did not produce a fluorescent response at any concentration of spermidine. The results are as follows... Figure 3 As shown.

[0144] Specificity detection: The spermidine fluorescent probes GRISPD-H and GRISPD-C were reactively detected with 14 similar substrates. The results showed that GRISPD-C had better specificity, while GRISPD-H could detect polyamines with a similar response amplitude, such as... Figure 4 As shown.

[0145] Dependence of GRISPD-C on calcium and magnesium ions: Spermine fluorescence probe GRISPD-C was detected using a concentration gradient (0–100 mM) of spermidine with or without calcium or magnesium ions. The change in the ratio of fluorescence intensity at 405 nm excitation to 550 nm emission and the ratio at 488 nm excitation to 550 nm emission were measured. The results showed that the response of GRISPD-C to spermidine was not affected by calcium or magnesium ions. Figure 5 As shown.

[0146] The effect of pH: For spermidine fluorescent probes GRISPD-H and GRISPD-C, spermidine was detected at concentration gradients (0–100 mM) in environments with pH values ​​of 6.0, 6.5, 7.0, 7.5, and 8.0. The changes in the ratio of fluorescence intensity at 405 nm excitation and 550 nm emission to fluorescence intensity at 488 nm excitation and 550 nm emission were measured. The results showed that the response amplitude of both GRISPD-H and GRISPD-C to spermidine was affected by pH. The Kd value of GRISPD-H to spermidine remained stable at different pH values, while the Kd value of GRISPD-C to spermidine was affected by pH to some extent. Figure 6 As shown.

[0147] The changes in fluorescence ratios of inactivated probes that have lost their spermidine responsiveness at different pH levels are similar to those of spermidine fluorescent probes. This allows for correction of the fluorescent probe's response at different pH levels, as shown in the following results. Figure 7 As shown.

[0148] Example 4: Subcellular organelle localization and intracellular performance of fluorescent probes

[0149] In this embodiment, different localization signal peptides are fused with the spermidine fluorescent probe GRISPD-C to localize the fluorescent probe to different organelles.

[0150] 293T cells were transfected with fluorescent probe plasmids fused with different localization signal peptides for 36 hours. After washing with PBS, the cells were placed in HBSS solution and fluorescence was detected under the GFP channel using a laser confocal fluorescence microscope. Results are as follows: Figure 8 As shown, the spermidine fluorescent probe GRISPD-C was fused with different subcellular localization signal peptides to obtain the fluorescent probe sequences shown in SEQ ID NO:13-18. These fluorescent probe sequences can be localized to subcellular structures such as the cytoplasm (SEQ ID NO:13), nucleus (SEQ ID NO:14), mitochondria (SEQ ID NO:15), Golgi apparatus (SEQ ID NO:16), endoplasmic reticulum (SEQ ID NO:17), and cell membrane (SEQ ID NO:18). Fluorescence was observed in all different subcellular structures, and the distribution and intensity of the fluorescence varied.

[0151] 293T cells were transfected with fluorescent probe plasmids expressing cytoplasm, nucleus, mitochondria, and Golgi apparatus for 36 hours. After washing with PBS, the cells were placed in HBSS solution and fluorescence was detected using a laser confocal microscope. Following the addition of 10 mM spermidine, the ratio of fluorescence intensity at 500-550 nm emission from 405 nm excitation to that at 500-550 nm emission from 488 nm excitation was continuously monitored. Results are as follows: Figure 9 As shown. The R of the sample with added spermidine. 488 / 405 As the fluorescence ratio gradually increases, the response amplitude of the probe varies in different organelles, reaching up to 3.2 times the initial value.

[0152] Example 5: Detection of spermidine concentration changes in subcellular compartments induced by drug treatment using fluorescent probes.

[0153] In this embodiment, the 293T cell line, stably expressing cytoplasmic, nuclear, Golgi apparatus, or mitochondrial localization probes, was used. After treating the cells for 16 hours with DFMO (an inhibitor of ornithine decarboxylase 1, a key enzyme in de novo polyamine synthesis) or N-(3-aminopropyl)cyclohexylamine (an inhibitor of spermine synthase 1, a downstream metabolic enzyme of spermine), the fluorescence ratio of the cells was detected by flow cytometry. The results are as follows: Figure 10 As shown, the probe can detect the decrease in spermidine concentration caused by efflunitine treatment and the increase in spermidine concentration caused by N-(3-aminopropyl)cyclohexylamine treatment in various organelles.

[0154] Example 6: Quantitative Detection of Spermine Concentration in Subcellular Compartments Using Fluorescent Probes

[0155] In this embodiment, a 293T cell line stably expressing cytoplasmic, nuclear, Golgi apparatus, or mitochondrial localization probes was used. Cells were treated with DFMO for 16 hours to obtain a spermidine-depleted cell model. Subsequently, the cell membrane was permeabilized with 0.0005% digitin for 30 min, and the intracellular spermidine concentration was equilibrated using different concentrations of spermidine. The R488 / 405 fluorescence ratio of the cells was detected by flow cytometry, and the kinetic curves of GRISPD-C response to spermidine in different subcellular structures were calculated. Figure 11 As shown.

[0156] By substituting the fluorescence ratio of the cells to be tested into the kinetic curve, the in situ physiological concentration of spermidine within the cells can be calculated. By substituting the fluorescence ratio of untreated cells into the corresponding kinetic curve, we calculated that the concentration of spermidine in the cytoplasm is approximately 220 μM, in the nucleus approximately 450 μM, in the Golgi apparatus approximately 500 μM, and highest in the mitochondria at approximately 650 μM.

[0157] Example 7: Application of fluorescent probes to verify the inhibitory effect of polyamine transport inhibitors

[0158] In this embodiment, the 293T cell line stably expressing the spermidine probe was used. Cells were placed in HBSS solution, and fluorescence detection was performed using laser confocal microscopy. After treatment with the polyamine transport inhibitor AMXT1501, 10 mM spermidine was added exogenously. The ratio of fluorescence intensity at 500-550 nm emission from 405 nm excitation to 500-550 nm emission from 488 nm excitation was continuously monitored to analyze the difference in spermidine uptake rate between the drug-treated group and the control group. The results are as follows: Figure 12 As shown, the application of spermidine probes can effectively verify the inhibitory effect of the transport inhibitor AMXT-1501.

[0159] Example 8: Detection of spermidine in blood using fluorescent probes

[0160] Nine two-month-old C57 male rats born at the same time were divided into three groups of three. The control group was provided with normal food and water. The starvation group was fasted from 6 PM to 10 AM the following morning (16 hours of fasting). The refeeding group was fasted from 6 PM to 8 AM the following morning, and then food was provided for 2 hours until 10 AM. Three 14-month-old C57 male rats were used as the aging group. All rats were euthanized at 10 AM the following morning, and blood samples were collected. After incubating the blood samples at 37°C for 2 hours, they were centrifuged at 4000 rpm and 4°C for 15 minutes to obtain serum. Serum was analyzed using a probe, and the differences in the fluorescence ratio of the probe among the different groups were compared. The results are as follows: Figure 13 As shown, the application of GRISPD-H can detect that the concentration of polyamines in the serum of aged mice is significantly lower than that in young mice, and the concentration of polyamines in the serum of young mice increases after fasting and refeeding.

[0161] As can be seen from the above embodiments, the spermidine fluorescent probe provided by the present invention has a relatively small protein molecular weight and is easy to mature. It exhibits large fluorescence dynamic changes and good specificity. Furthermore, it can be expressed in cells through gene manipulation, enabling highly specific, high-throughput, real-time, and quantitative dynamic detection of spermidine both intracellularly and in vitro.

Claims

1. A spermidine fluorescent probe, characterized in that, This includes spermidine-sensitive peptides and optically active peptides.

2. The spermidine fluorescent probe according to claim 1, characterized in that, The spermidine-sensitive polypeptide is any one of spermidine-binding protein, spermidine-binding protein functional variant 1, spermidine-binding protein functional variant 2, and spermidine-binding protein functional variant 3; the spermidine-sensitive polypeptide is derived from Pseudomonas aeruginosa; the amino acid sequence of the spermidine-binding protein is shown in SEQ ID NO:1; The spermidine-binding protein functional variant 1 is obtained by mutation of the spermidine-binding protein; the mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion. The mutation sites are any 1 to 5 of the following: T35, D36, P81, T134, V141, V147, P152, A157, E161, D180, E181, A185, N195, H197, K200, K203, F219, K223, Y239, D242, N269, W271, D273, P297, A301, Y309, L319, and Y331.

3. The spermidine fluorescent probe according to claim 2, characterized in that, The mutations are any of the following combinations: F219 and N269; P81, F219 and N269; V141, F219 and N269; F219, K223 and N269; E181, F219, K223 and N269; T134, E161, F219 and N269; F219, N269, L319 and Y331; D180, F219, N269, L319 and Y331; T134, E161, D180, F219 and N269; T134, F219, N269 and L319; K200, F219, N269 and L319; E161, A185, F219 and N269; The mutations are as follows: P81 mutates to Q, T134 mutates to I, E161 mutates to I, D180 mutates to A, E181 mutates to A, A185 mutates to M, K200 mutates to I, F219 mutates to W or D, K223 mutates to Q, N269 mutates to G or P, L319 mutates to D or E, and Y331 mutates to G.

4. The spermidine fluorescent probe according to claim 3, characterized in that, The mutations are any of the following combinations: F219D and N269G; F219W and N269G; P81Q, F219D and N269G; F219W, K223Q and N269G; E181A, F219W, K223Q and N269G; T134I, E161I, F219W and N269G; F219W, N269G, L319D and Y331G; D18 0A, F219W, N269G, L319D and Y331G; T134I, E161I, D180A, F219W and N269G; T134I, F219W, N269G and L319D; K200I, F219W, N269G and L319D; E161I, A185M, F219W and N269G; V141L, F219W and N269G.

5. The spermidine fluorescent probe according to claim 4, characterized in that, The spermidine-binding protein functional variant 2 is a truncated variant of amino acids 28 to 362 of the amino acid sequence of spermidine-binding protein functional variant 1. The spermidine-binding protein functional variant 3 is a nucleotide sequence that has ≥70% sequence similarity to spermidine-binding protein functional variant 1.

6. The spermidine fluorescent probe according to claim 1, characterized in that, The optically active polypeptide is located between residues 67-72, 218-224, or 337-342 of the spermidine-sensitive polypeptide; The optically active polypeptide is any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and any one of the sequences containing 1 to 6 mutations at any of the Y1, N2, D4, K22, I27, F59, K62, S130, Y139, F199, and N205 sites in SEQ ID NO:2; The mutation is any one of amino acid modification, amino acid substitution, and amino acid deletion.

7. The spermidine fluorescent probe according to claim 6, characterized in that, The mutation is any one or more of the following: Y1G, N2G, D4H, K22W, I27V, F59Y, K62A, S130R, Y139N, F199T and N205F; The optically active polypeptide is located at one or more of the following sites on the spermidine-sensitive polypeptide: 67 / 68, 67 / 69, 67 / 70, 67 / 71, 67 / 72, 68 / 69, 68 / 70, 68 / 71, 68 / 72, 69 / 70, 69 / 71, 69 / 72, 70 / 71, 70 / 72, 71 / 72, 218 / 219, 218 / 220, 218 / 221, 218 / 222, 218 / 223, 218 / 224, 219 / 220, 219 / 221, 219 / 222, 219 / 223. 219 / 224, 220 / 221, 220 / 222, 220 / 223, 220 / 224, 221 / 222, 221 / 223, 221 / 224, 222 / 223, 222 / 224, 223 / 224, 337 / 338, 337 / 339, 337 / 340, 337 / 341, 337 / 342, 338 / 339, 338 / 340, 338 / 341, 338 / 342, 339 / 340, 339 / 341, 339 / 342, 340 / 341, 341 / 342.

8. The spermidine fluorescent probe according to claim 7, characterized in that, The amino acid sequence of the spermidine fluorescent probe is any one of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:

18.

9. The nucleotide sequence encoding the spermidine fluorescent probe according to any one of claims 1 to 8.

10. The application of the spermidine fluorescent probe according to any one of claims 1 to 8 or the nucleotide sequence encoding the spermidine fluorescent probe according to claim 9 in the localization or quantitative detection of spermidine.

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