A fusion protein, 2-ketoglutaric acid biosensor and its application

Abstract

This invention belongs to the field of biosensor technology, specifically relating to a fusion protein, a 2-ketoglutaric acid biosensor, and its applications. Specifically, this invention combines the MifS extracellular sensing domain MifS-SD of the MifS / MifR two-component signal transduction system in Pseudomonas aeruginosa PAO1 with a superfolded circular rearranged yellow fluorescent protein, successfully constructing a 2-ketoglutaric acid biosensor with a high response amplitude. Experiments demonstrate that this biosensor can achieve real-time, in-situ detection of 2-ketoglutaric acid in microorganisms and mammalian cells, and can support the detection of related enzyme activities and the screening of inhibitors in a highly efficient, low-cost, and high-throughput manner, thus possessing good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biosensing technology, specifically relating to a fusion protein, 2-ketoglutaric acid biosensor and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] 2-Ketoglutarate (2-KG), also known as 2-oxoglutarate (2-OG), is a core metabolic intermediate in the tricarboxylic acid cycle (TCA cycle), widely involved in various cellular metabolic pathways such as nitrogen assimilation, amino acid synthesis, and energy metabolism. Furthermore, 2-KG functions as a signaling molecule or regulator, playing a crucial role in key biological processes such as hypoxia response, epigenetic modification, inflammatory responses, and cell fate determination. Its metabolic disorders are closely related to the development and progression of various metabolic syndromes, including obesity, diabetes, and cardiovascular disease.

[0004] However, 2-KG itself lacks fluorescence and characteristic absorption peaks, making direct detection of its concentration to reflect the activity of related metabolic enzymes extremely technically challenging. Currently used enzyme activity detection methods include high-performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (1H-MRS). While these methods can accurately measure 2-KG, they generally suffer from cumbersome operation, reliance on expensive equipment, and low throughput, making it difficult to achieve rapid, convenient, and high-throughput enzyme activity analysis. Although some enzyme-coupled methods or bioluminescent or fluorescent probe-based technologies have been used for high-performance mutant screening or inhibitor screening of specific 2-KG-related enzymes, a universal, direct method applicable to a wide range of in vitro and in vivo scenarios for 2-KG-related enzyme activity detection is still lacking.

[0005] Furthermore, traditional methods are limited by their invasiveness, low spatial resolution, and inability to perform real-time dynamic detection, making it difficult to effectively track dynamic changes in 2-KG metabolism in live cell and in vivo studies. Although various gene-encoded fluorescent biosensors, such as P... II -TC3、P II NAGK, Nifa-FRET, OGsor, mOGsor, etc. have been developed for 2-KG metabolism research, but they have problems such as small dynamic range and insufficient sensitivity to subtle 2-KG fluctuations, which limits the in-depth metabolic and functional research under physiological and pathological conditions. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, the inventors, through long-term technical and practical exploration, have provided a fusion protein, a 2-ketoglutaric acid biosensor, and its applications. Specifically, the 2-KG biosensor comprises at least a fusion protein consisting of the extracellular sensing domain of MifS in the MifS / MifR two-component signal transduction system and a cyclic rearranged fluorescent protein. Experiments have demonstrated that the biosensor developed in this invention possesses advantages such as high response amplitude, strong fluorescence intensity, moderate affinity, and ease of operation, enabling efficient, high spatiotemporal resolution, and low-cost qualitative or quantitative detection of intracellular and extracellular 2-KG. Based on the above research results, this invention has been completed.

[0007] Specifically, to achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a fusion protein comprising an extracellular sensing domain of MifS and a fluorescent protein; wherein the extracellular sensing domain of MifS may be derived from Pseudomonas aeruginosa (…). Pseudomonas aeruginosa The MifS extracellular sensor domain (SD) in the MifS / MifR two-component signal transduction system of PAO1 has been found to bind to 2-KG; the fluorescent protein may be a cyclic rearranged fluorescent protein.

[0009] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned fusion protein.

[0010] A third aspect of the present invention provides a recombinant expression vector containing the isolated nucleic acid molecules described above.

[0011] In a fourth aspect, the present invention provides a host cell containing the isolated nucleic acid molecule, the recombinant expression vector, or a fusion protein expressed as described above.

[0012] A fifth aspect of the present invention provides a method for preparing the above-described fusion protein, the method comprising: The host cells were cultured, and the fusion protein was isolated and purified to obtain the fusion protein.

[0013] A sixth aspect of the present invention provides the use of the above-described fusion protein, nucleic acid molecule, vector and / or host cell in the preparation of a 2-KG biosensor.

[0014] A seventh aspect of the present invention provides a 2-KG biosensor, the 2-KG biosensor comprising at least the above-described fusion protein.

[0015] An eighth aspect of the present invention provides a method for in vitro detection of 2-KG, the method comprising at least: contacting a sample to be tested with the 2-KG biosensor, and analyzing the concentration or presence of 2-KG in the sample to be tested based on changes in the fluorescence signal of the biosensor.

[0016] A ninth aspect of the present invention provides a method for detecting 2-KG in vivo (cells), the method comprising at least: inducing the expression of the biosensor in cells, and analyzing the concentration or presence of 2-KG in cells based on changes in the fluorescence signal of the biosensor.

[0017] A tenth aspect of the present invention provides a method for detecting the activity of 2-KG-related enzymes or for high-throughput screening of 2-KG-related enzyme materials, the method comprising: contacting a test sample containing or suspected of containing 2-KG-related enzymes with the biosensor, and analyzing the activity of the 2-KG-related enzymes to be tested based on changes in the fluorescence signal of the biosensor.

[0018] The 2-KG-related enzyme materials include 2-KG-related enzyme inhibitors or 2-KG-related enzyme promoters. No specific limitations are specified here.

[0019] In an eleventh aspect of the present invention, the application of the above-described fusion protein, 2-KG biosensor, and / or the above-described detection method in the detection of 2-KG-related diseases and / or the screening of related drugs is provided.

[0020] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects: (1) The 2-KG biosensor provided by the above technical solution is constructed by inserting the hyperfolded circular rearranged fluorescent protein sfcpYFP into an appropriate position inside the extracellular sensing domain (MifS-SD) of the MifS / MifR two-component signal transduction system derived from Pseudomonas aeruginosa PAO1. The conformational change induced by the binding of 2-KG to MifS-SD leads to conformational changes in the polypeptide backbone and amino acid side chains at the insertion site of the circular rearranged fluorescent protein, which further significantly changes the fluorescence intensity of the circular rearranged fluorescent protein. This change in fluorescence intensity can be used as a detection index for 2-KG concentration.

[0021] (2) The 2-KG biosensor provided by the above technical solution (named FiKG, amino acid sequence as shown in SEQ ID NO.1, nucleotide sequence as shown in SEQ ID NO.2) has a response amplitude of 1620.24 ±24.36% to 2-KG, a detection range of 0.017-2.39 mM, and a detection limit as low as 1.51 μM. It has high specificity, temperature stability and reversible binding to 2-KG.

[0022] (3) The 2-KG biosensor FiKG provided by the above technical solution can be expressed in bacterial and mammalian cells, thereby enabling in situ and real-time detection of 2-KG in microorganisms and mammalian cells, which is of great significance for the development of research on the physiological function diversity of 2-KG.

[0023] (4) The 2-KG biosensor FiKG provided by the above technical solution offers an efficient, convenient, and low-cost method for detecting enzyme activity in enzymes that use 2-KG as a substrate or product. By adding purified FiKG protein to the enzyme activity detection system and combining it with real-time detection of FiKG fluorescence changes using a fluorescence microplate reader, the activity of the corresponding enzyme can be rapidly and sensitively determined.

[0024] (5) The above technical solutions further expand the application value of sensors in enzyme engineering and drug screening. By combining the 2-KG biosensor FiKG with a fluorescent microplate reader and multi-well plates (such as 96-well or 384-well plates), high-performance mutants of 2-KG-related enzymes and high-throughput screening of enzyme inhibitors can be achieved. Only by adding purified FiKG protein and the target compound to a unified detection system can the activity changes or inhibitory effects of 2-KG-related enzymes be evaluated in parallel and rapidly, thus demonstrating good practical application value. Attached Figure Description

[0025] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 This demonstrates the specificity of the fluorescence thermal migration experiment used to analyze MifS-SD in Example 1 of this invention.

[0027] Figure 2 This is a schematic diagram of the structure of different 2-KG biosensor variants formed by the superfolded circular rearranged fluorescent protein sfcpYFP screened in Example 1 of the present invention at different insertion sites inside MifS-SD, and a comparison of the response amplitude to 2-KG.

[0028] Figure 3 FiKG in Embodiment 1 of the present invention 0.1 Schematic diagram of the structure and dose-response curve for 2-KG.

[0029] Figure 4 The 672 FiKGs selected in Example 1 of this invention 0.1 Comparison of the response magnitude of linker random mutants to 2-KG.

[0030] Figure 5This is a schematic diagram of the structure of FiKG in Embodiment 1 of the present invention and its dose-response curve for 2-KG.

[0031] Figure 6 In Embodiment 2 of the present invention E. coli Gradient response and specificity analysis of FiKG expressed in W3110 to 2-KG. A is E. coli The fluorescence ratio of FiKG expressed in W3110 to gradient concentrations of 2-KG; B is... E. coli Specificity analysis of FiKG expressed in W3110.

[0032] Figure 7 The FiKG-based method in Embodiment 2 of the present invention E. coli Functional verification of 2-KG transporter, synthesizer, and catabolite enzymes in W3110. A is... E. coli W3110 and E. coli W3110- kgtP The expression in B represents the real-time response of FiKG to an externally added 2-KG; B is... E. coli W3110 E. coli W3110- icdA , E. coli W3110- sucA , E. coli W3110- ydiJ , E. coli W3110- serA and E. coli W3110- ydiJ serA Comparison of fluorescence ratios of FiKG expressed in the medium.

[0033] Figure 8 This is the real-time response of FiKG to the addition of exogenous 2-KG in human embryonic kidney cells HEK293FT in Example 3 of the present invention.

[0034] Figure 9 This image shows the localization of FiKG in different subcellular compartments of human embryonic kidney cells HEK293FT and its response to 2-KG in Example 3 of the present invention. A is an imaging result of the localization of FiKG in different subcellular compartments of human embryonic kidney cells HEK293FT; B is the response of FiKG to 2-KG in different subcellular compartments.

[0035] Figure 10This is the FiKG-based L-glutamate oxidase activity assay in Example 4 of the present invention. A shows the time progression curve of 2-KG generation in the L-glutamate oxidase catalytic system determined by FiKG; B shows a comparison of L-glutamate oxidase activity determined by FiKG and by the horseradish peroxidase-coupled method.

[0036] Figure 11 This is the FiKG-based aspartate aminotransferase activity assay in Example 4 of the present invention. A shows the time progression curve of 2-KG consumption in the aspartate aminotransferase catalytic system determined by FiKG; B shows a comparison of aspartate aminotransferase activity determined by FiKG and by the coupled malate dehydrogenase method.

[0037] Figure 12 This is the FiKG-based isoleucine dioxygenase activity detection in Example 4 of the present invention. A shows the time progression curve of 2-KG consumption in the isoleucine dioxygenase catalytic system determined by FiKG; B shows the time progression curve of FiKG and (2-KG) consumption in the isoleucine dioxygenase catalytic system. S ,3 R 4 S Comparison of isoleucine dioxygenase activities determined by the 4-hydroxyisoleucine dehydrogenase coupling method.

[0038] Figure 13 This is an analysis of the differences in the activity of FiKG-based co-expressed isoleucine dioxygenase in Example 4 of the present invention.

[0039] Figure 14 This refers to the proline hydroxylase 2 inhibitor in the compound library selected from the FiKG two-round high-throughput screening in Example 5 of this invention.

[0040] Figure 15 This is a FiKG-based determination of the half-maximal inhibitory concentration (IC50) of varadastatin and Evans blue against proline hydroxylase 2 in Example 5 of the present invention. A shows the FiKG inhibition curve of varadastatin against proline hydroxylase 2; B shows the FiKG inhibition curve of Evans blue against proline hydroxylase 2. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0042] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0043] Terminology Definition In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein, such as those in virology, biochemistry, nucleic acid chemistry, and immunology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0044] In this invention, "fusion protein" refers to oligopeptides, peptides, protein sequences or fragments thereof, and specifically to molecules that are actually formed, recombinant, synthetic, or semi-synthetic. It should be noted that the term "fusion protein" and similar terms do not imply that the amino acid sequence is limited to the full-length molecule containing the complete natural amino acid sequence. It should be understood that the various references to "fusion protein" and similar terms in this invention will include full-length sequences as well as any fragments, derivatives, or variants thereof.

[0045] In this invention, a "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of this invention, preferably, a nucleic acid molecule is a DNA fragment. A "nucleic acid molecule" is also called a polynucleotide molecule.

[0046] In this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0047] In this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0048] In this invention, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of compared positions multiplied by 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match).

[0049] In this invention, the twenty common amino acids involved are written in accordance with conventional usage. For example, Immunology-ASynthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0050] In a typical embodiment of the present invention, a fusion protein is provided, which is composed of a MifS extracellular sensing domain and a cyclic rearranged fluorescent protein.

[0051] The extracellular sensing domain of MifS can be derived from Pseudomonas aeruginosa (…). Pseudomonas aeruginosa The extracellular sensor domain (SD) of MifS in the PAO1 MifS / MifR two-component signal transduction system has been found to bind to 2-KG. Of course, based on the concept of this invention, other existing two-component signal transduction systems that can recognize 2-KG extracellular sensor domains are also applicable to the technical solutions of this application and should therefore fall within the scope of protection of this application.

[0052] The circular rearranged fluorescent proteins are a class of visual reporter gene-encoded proteins, including circular rearranged cyan fluorescent protein (cpTFP), circular rearranged green fluorescent protein (cpGFP), circular rearranged yellow fluorescent protein (cpYFP), and circular rearranged red fluorescent protein (cpRFP). In one specific embodiment of the present invention, the circular rearranged fluorescent protein used is the hyperfolded circular rearranged yellow fluorescent protein sfcpYFP. When 2-KG is present, the conformational change of MifS-SD induced by the binding of 2-KG to the extracellular sensing domain MifS-SD of MifS can lead to a conformational change of sfcpYFP, ​​thereby greatly altering the fluorescence properties of the sensor and enabling the detection of 2-KG.

[0053] Furthermore, the fusion protein can be obtained by linking the cyclic rearranged fluorescent protein to the amino terminus or carboxyl terminus of the extracellular sensing domain of MifS, or the fusion protein can be obtained by inserting the cyclic rearranged fluorescent protein into the extracellular sensing domain of MifS. Specifically, the amino acid sites of the cyclic rearranged fluorescent protein sfcpYFP inserted into MifS-SD are as follows: 41A / 42L, 42L / 43D, 43D / 44P, 51A / 52G, 52G / 53P, 53P / 54I, 54I / 55D, 55D / 56G, 140R / 141A, 141A / 142E, 142E / 143D, 143D / 144G, 144G / 145S, 145S / 146F, 211R / 212Q, 212Q / 213P, 2 13P / 214L, 214L / 215T, 215T / 216P, 216P / 217L, 217L / 218R, 218R / 219H, 224S / 225Y, 225Y / 226G, 226G / 227E, 227E / 228D, 228D / 229R, 229R / 230R, 230R / 231L, 249D / 250L, 250L / 251P, 251P / 252N, 252N / 253D, 253D / 254G, 254G / 255W, 261R / 262D, 262D / 263T, 263T / 264A, 264A / 265S, 265S / 266I, 266I / 267Q, 267Q / 268D, 268D / 269D, 269D / 270V. It should be noted that sfcpYFP are linked to the upper and lower fragments of MifS-SD via linkers with N-terminal "serine-alanine-glycine" and C-terminal "glycine-glycine-cysteine," respectively.

[0054] Specifically, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

[0055] In (a2), the substitution, deletion and / or addition of one or more amino acid residues generally refers to the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0056] In one specific embodiment of the invention, the fusion protein further comprises a signal peptide and / or a tag protein. Further, the fusion protein comprises a signal peptide at its N-terminus. In some embodiments, the fusion protein comprises a tag protein, such as a His tag, at its C-terminus. No specific limitations are imposed herein.

[0057] The fusion proteins described in this invention can be prepared using various methods known in the art, such as genetic engineering (recombinant technology) or chemical synthesis (e.g., the Fmoc solid-phase method). The fusion proteins of this invention are not limited by their method of preparation.

[0058] In one or more embodiments of the present invention, an isolated nucleic acid molecule is provided, the nucleic acid molecule being capable of encoding the aforementioned fusion protein.

[0059] Specifically, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)-(b3) and encode a fusion protein with the same function.

[0060] In the above nucleic acid molecules, the stringent conditions can be as follows: hybridization at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 2×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 1×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in 0.5×SSC and 0.1% SDS; or hybridization at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 50 °C in a mixed solution of 0.5 M Na3PO4 and 1 mM EDTA. Alternatively, hybridization can be performed at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, followed by rinsing at 65 °C in 0.1×SSC and 0.1% SDS; or hybridization can be performed at 65 °C in a solution of 6×SSC and 0.5% SDS, followed by washing once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0061] It should be noted that the term "identity" refers to sequence similarity to an amino acid / nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0062] The aforementioned 40% or more of identity can be 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% or more of identity.

[0063] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., without specific limitations.

[0064] In one or more specific embodiments of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector comprises at least the above-mentioned nucleic acid molecules.

[0065] The recombinant expression vector can be any one or more of a viral vector, plasmid, bacteriophage, phage particle, granule, or artificial chromosome; the viral vector may include adenovirus vector, retrovirus vector, or adeno-associated virus vector; the artificial chromosome includes bacterial artificial chromosome, phage P1-derived vector, yeast artificial chromosome, or mammalian artificial chromosome; more preferably, it is a plasmid; the plasmid includes, but is not limited to, pET28a and pcDNA3.1. (+) .

[0066] In one or more specific embodiments of the present invention, a host cell is provided, wherein the host cell contains the above-mentioned nucleic acid molecule, the above-mentioned recombinant expression vector, or is capable of expressing the above-mentioned fusion protein.

[0067] The host cells include bacterial cells, fungal cells, and animal cells; The bacteria can be any one or more of Escherichia coli, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus.

[0068] In one or more specific embodiments of the present invention, the bacteria include, but are not limited to, Escherichia coli (e.g., BL21(DE3)), Agrobacterium tumefaciens (e.g., GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus cereus, or Pseudomonas fluorescens.

[0069] The fungal cells include yeast.

[0070] The animal cells may be mammalian cells, and more specifically, the mammalian cells include, but are not limited to, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293FT cells.

[0071] In one or more specific embodiments of the present invention, a method for preparing the above-mentioned fusion protein is provided, the method comprising: culturing the above-mentioned host cells, and isolating and purifying the fusion protein.

[0072] In one or more specific embodiments of the present invention, the application of the above-mentioned fusion protein, nucleic acid molecule, recombinant expression vector and / or host cell in the preparation of a biosensor for detecting 2-KG is provided.

[0073] In one or more specific embodiments of the present invention, a biosensor for detecting 2-KG is provided, wherein the biosensor comprises at least the above-mentioned fusion protein.

[0074] The 2-KG biosensor can be a FiKG biosensor constructed by inserting sfcpYFP with an N-terminal linker of "alanine" and a C-terminal linker of "leucine-proline" between amino acids 29-215 and 216-298 of MifS-SD (its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2).

[0075] Furthermore, the biosensor may also include other reagents, devices, and / or equipment for 2-KG detection.

[0076] For example, in this invention, the reagent may contain a detection buffer (such as fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0077] In practical applications, the aforementioned biosensors can exist in the form of detection kits, especially for 2-KG detection in (in vitro) environments.

[0078] When biosensors are used to detect 2-KG in the body (such as in cells), they can be directly induced to express in cells, thereby detecting the presence or concentration of intracellular 2-KG.

[0079] Therefore, in one or more specific embodiments of the present invention, a method for in vitro detection of 2-KG is provided, the method comprising at least: contacting the sample to be tested with the biosensor, and analyzing the concentration or presence of 2-KG in the sample to be tested based on the change in fluorescence signal of the 2-KG biosensor.

[0080] The sample to be tested is a sample containing or suspected of containing 2-KG. The sample can be a biological sample or an environmental sample. The biological sample includes, but is not limited to, bacterial culture medium, bacterial lysate, cell culture medium, cell lysate, animal blood (such as whole blood, serum, dried blood spot samples, etc.), animal urine, and animal tissue fluid. The animal may be a mammal, with humans being preferred.

[0081] The environmental samples include, but are not limited to, water bodies (such as surface water and wastewater), soil leachate, food processing liquids, and fermentation liquids. Optionally, the samples may be pretreated, such as by dilution, filtration, centrifugation to remove impurities, and adjustment of pH or ionic strength, to adapt them to the detection environment of the biosensor.

[0082] At this time, the 2-KG may also include other reagents, devices and / or equipment for 2-KG detection; The reagents include detection buffers (such as fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4).

[0083] In one or more specific embodiments of the present invention, a method for intracellular detection of 2-KG is provided, the method comprising at least: inducing the expression of a 2-KG biosensor in cells, and analyzing the concentration or presence of 2-KG in cells based on changes in the fluorescence signal of the 2-KG biosensor.

[0084] At this point, the 2-KG biosensor is the aforementioned fusion protein.

[0085] The cells may be bacterial cells, fungal cells, or animal cells, and are not specifically limited thereto. In specific embodiments of the present invention, the cells include, but are not limited to, Escherichia coli W3110 cells and human embryonic kidney cells (HEK293FT).

[0086] The above methods can be used to achieve qualitative or quantitative detection of 2-KG in vitro or in vivo.

[0087] In another specific embodiment of the present invention, a method for detecting the activity of 2-KG-related enzymes or for high-throughput screening of 2-KG-related enzyme materials is provided. The method includes: contacting a test sample containing or suspected of containing 2-KG-related enzymes with the biosensor, and analyzing the activity of the 2-KG-related enzymes to be tested based on changes in the fluorescence signal of the biosensor.

[0088] The 2-KG-related enzymes include, but are not limited to, those derived from Streptomyces ( Streptomyces L-glutamate oxidase (l-GOX) of sp.) X-119-6, Corynebacterium glutamicum (sp.) Corynebacterium glutamicum ATCC 13032 aspartate aminotransferase (AspB), Bacillus subtilis ( Bacillus subtilis The 2-KG-related enzymes include isoleucine dioxygenase IDO (K2I17) and human proline hydroxylase 2 (PHD2) and its mutants. Furthermore, the 2-KG-related enzyme can be human proline hydroxylase 2 (PHD2). 181-426 ).

[0089] The 2-KG-related enzyme materials include 2-KG-related enzyme inhibitors or 2-KG-related enzyme promoters. No specific limitations are specified here.

[0090] In another specific embodiment of the present invention, the application of the above-mentioned fusion protein, 2-KG biosensor and / or the above-mentioned detection method is provided in the detection of 2-KG-related diseases and / or the screening of related drugs.

[0091] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. In the following embodiments, the expression vectors pETDuet-1 and pcDNA3.1 are used. (+) All materials and reagents used were purchased from Novagen; unless otherwise specified, all other materials and reagents used were obtained commercially. Unless otherwise specified, all experimental methods used were standard methods.

[0092] Example 1: Construction and optimization of a 2-KG biosensor The culture medium and reagents used in this embodiment are as follows: LB medium: 0.5% yeast extract, 1% peptone, 1% NaCl; PBS buffer (1 L): 19.1 g Na2HPO4·12H2O, 1.82 g KH2PO4; Binding buffer: 20 mM Na2HPO4, 20 mM imidazole, 500 mM NaCl, pH 7.4; Elution buffer: 20 mM Na2HPO4, 500 mM imidazole, 500 mM NaCl, pH 7.4; Fluorescence thermal migration assay reaction buffer: 20 mM Na2HPO4, 150 mM NaCl, pH 7.4; Fluorescence assay buffer: 50 mM Tris-HCl, pH 7.4.

[0093] (1) Specificity analysis of the extracellular sensory domain of MifS PCR amplification was derived from Pseudomonas aeruginosa ( Pseudomonas aeruginosa The gene encoding the MifS extracellular sensory domain (MifS-SD) in PAO1 mifS 29-298 (NC_002516.2), using EcoR I / Hind The gene fragment was digested with enzyme III and ligated with the pET28a plasmid using T4 DNA ligase to obtain the recombinant plasmid pET28a-MifS-SD, which was then transformed into the expression strain. E. coli In BL21(DE3), strains that were successfully constructed were screened by plating on LB agar plates containing kanamycin sulfate. Single colonies were picked, and the strains were preserved after successful PCR verification of the bacterial culture.

[0094] After activating the verified single clones in LB medium for two generations, they were inoculated at a 2% inoculum into 500 mL of LB medium containing kanamycin sulfate resistance (50 μg / mL) and cultured at 37°C with shaking at 180 rpm until OD. 600 When the pH reaches 0.6-0.8, add 1 mM IPTG to the culture medium and induce induction at 23°C and 160 rpm for 14 hours; collect the bacterial cells by centrifugation at 6,000 rpm for 10 minutes, wash the bacterial cells twice with binding buffer, and resuspend to OD. 600 Add 10% glycerol and 1 mM PMSF to a concentration of 20. Hypolyte the cells using a high-pressure homogenizer at 800 Pa, repeating the process four times. Centrifuge the homogenate at 4°C and 12,000 rpm for 50 minutes to remove cell debris. Filter the supernatant through a 0.22 μm filter and purify it using a 5 mL nickel column. Elute with different concentrations of elution buffer to obtain purified MifS-SD.

[0095] To determine that 2-KG is the specific ligand for MifS-SD, purified MifS-SD was reacted with 1 mM 2-KG, glycolysis and tricarboxylic acid cycle intermediates, amino acids, ATP or ADP, and Mg. 2+ The metal ions were mixed separately for fluorescence thermal migration experiments, and the thermal stability of MifS-SD was analyzed using a LightCycler 480 system for quantitative PCR. Figure 1 ).

[0096] The fluorescence thermal migration assay was performed using SYPRO orange dye. The quantitative PCR instrument was set to an excitation wavelength of 465 nm and an emission wavelength of 580 nm. Each 25 μL reaction mixture contained 8 μM MifS-SD, 5 × SYPRO orange, 1 mM of different compounds, and reaction buffer. All reactions were performed in white 96-well plates, with the temperature increasing by 1.2 °C / second per cycle within the range of 25–95 °C. The melting temperature (T0) of MifS-SD was calculated based on the negative first derivative of the raw fluorescence data. m Thermal migration temperature (ΔT) m T by adding different compounds to MifS-SD m Subtract the control T m Calculate ΔT m Compounds with temperatures above 2 °C may be potential ligands for MifS-SD.

[0097] PCR amplification Pseudomonas aeruginosa PAO1 origin mifS 29-298 The gene and primer design are as follows: Upstream primer: 5'-AAATGGGTCGCGGATCC GAATTC CGCCAGGCGCGGCAGCA -3', carrying a EcoR I site; Downstream primer: 5'-CGAGTGCGGCCGC AAGCTT CTAGACGTCGTCCTGGATG -3', carrying a Hind Site III.

[0098] (2) Construction of 2-KG biosensor In this embodiment, based on pET28a-MifS-SD, the following sites are selected for insertion of sfcpYFP to construct pET28a-FiKG. X / Y (In this article, FiKG) X / Y This is represented as a sensor formed by inserting sfcpYFP between the X and Y amino acids in the MifS-SD sequence; such as FiKG.215T / 216P Recombinant plasmids for inserting cpYFP between amino acids 215T and 216P in the MifS-SD sequence: 41A / 42L, 42L / 43D, 43D / 44P, 51A / 52G, 52G / 53P, 53P / 54I, 54I / 55D, 55D / 56G, 140R / 141A, 141A / 142E, 142E / 143D, 143D / 144G, 144G / 145S, 145S / 146F, 211R / 212Q, 212Q / 213P, 213P / 214L, 214L / 215T, 215T / 216P, 216P / 217L, 217L / 218R, 218R / 2 Forty-four sensor insertion variant encoding plasmids were constructed, including 19H, 224S / 225Y, 225Y / 226G, 226G / 227E, 227E / 228D, 228D / 229R, 229R / 230R, 230R / 231L, 249D / 250L, 250L / 251P, 251P / 252N, 252N / 253D, 253D / 254G, 254G / 255W, 261R / 262D, 262D / 263T, 263T / 264A, 264A / 265S, 265S / 266I, 266I / 267Q, 267Q / 268D, 268D / 269D, and 269D / 270V. In the recombinant plasmids encoding the various sensor variants, sfcpYFP is linked to the upper and lower segments of MifS-SD by linkers with N-terminal "serine-alanine-glycine" and C-terminal "glycine-glycine-cysteine", respectively.

[0099] The 44 sensor insertion variant coding plasmids were transformed into *E. coli* BL21(DE3) and screened for successful strains on LB agar plates containing kanamycin sulfate. Single colonies exhibiting bright fluorescence were picked and placed in 1.5 mL centrifuge tubes, which were then inoculated into 50 mL centrifuge tubes containing 15 mL of LB and cultured at 37°C with shaking at 180 rpm until OD500. 600When the pH was 0.6-0.8, 1 mM IPTG was added to the culture medium, and the cells were induced at 23°C and 160 rpm for 14 hours. The cells were collected by centrifugation at 6,000 rpm for 10 minutes, washed and resuspended with PBS buffer, and transferred to 48-well deep-well plates. 1 mM PMSF was added. The cells were disrupted using a Scientz-48TD multichannel ultrasonic disruptor, and 1 mL of the disruption was centrifuged at 4°C and 13,000 rpm for 10 minutes. The supernatant was collected. The supernatant was mixed with 0 and 1 mM 2-KG, and 100 μL of the mixture was transferred to a black 96-well plate. The fluorescence intensity was detected using an Ensight microplate reader. The instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, and emission wavelength of 528 nm. The ratio of the fluorescence intensity measured at 488 nm to that measured at 405 nm for each insert variant was calculated. R (F) 488 nm / F 405 nm ), and selected the insert variants with high response amplitude, and performed exogenous expression and purification according to the method in (1). Figure 2 ).

[0100] The purified sensor variant proteins and gradient concentrations of 2-KG were diluted separately using detection buffer. The purified sensor and 2-KG solution were mixed at a volume ratio of 3:1. 100 μL of the mixture was transferred to a black 96-well plate, and the fluorescence intensity was detected using an Ensight fluorescence microplate reader. The fluorescence ratio was calculated. R Plotting 2-KG concentration on the x-axis, F 488 nm / F 405 nm Using GraphPad Prism 8.0 as the ordinate, the dose-response curve of the sensor to 2 kg was obtained by fitting the curve, and the maximum response amplitude of the sensor to 2 kg was calculated. R max (i.e., the maximum ratio change). For example... Figure 3 As shown, the sensor variant obtained by inserting sfcpYFP into the MifS-SD at amino acid sites 215T / 216P... R max The maximum was 163.12 ± 7.87%. K d The value was 928.5 ± 88.08 μM, and it was named FiKG. 0.1 .

[0101] pET28a-FiKG 41A / 42L to pET28a-FiKG 269D / 270VThe sensor gene fragments were constructed as follows: using recombinant plasmid pET28a-MifS-SD as a template, the linearized plasmid backbone broken at different insertion sites in MifS-SD was amplified by reverse PCR; using recombinant plasmid pET28a-sfcpYFP as a template, the sfcpYFP fragment was amplified by PCR, and the sfcpYFP fragment and the linearized plasmid backbone were recombined using a DNA assembly method based on T5 exonuclease to obtain recombinant plasmids for each sensor insert variant.

[0102] The formulation of the 15 μL ligation system in the DNA assembly method based on T5 exonuclease described in (1) above is as follows: 4 μL 5× isothermal reaction buffer (0.5 M Tris-HCl, 0.05 M MgCl2, 0.05 M dithiothreitol), 0.004 μL 10 U μL -1 T5 exonuclease, 11 μL ddH2O.

[0103] PCR amplification Pseudomonas aeruginosa The primer design is as follows: The PAO1-derived fragment from MifS-SD and the partially linearized plasmid backbone are used: MifS-SD fragment 41A / 42L and the downstream primer of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTCGCCAGTACCGCC-3'; MifS-SD fragment 42L / 43D and downstream primers of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTCAGCGCCAGTACCG-3'; MifS-SD fragment 43D / 44P and downstream primers of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTATCCAGCGCCAGTA-3'; MifS-SD fragment 51A / 52G and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGGCCAGCGCCGCGCG-3'; MifS-SD fragment 52G / 53P and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTCCCGGCCAGCGCCGCG-3'; MifS-SD fragment 53P / 54I and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTCGGCCCGGCCAGCGCCGC-3'; MifS-SD fragment 54I / 55D and downstream primers of the partially linearized plasmid backbone: 5'-TCGCTGTTGTAGCCTGCAGAGCGTTTGTCGAGCATCTCC-3'; MifS-SD fragment 55D / 56G and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCGATCGGCCCGGCCA-3'; MifS-SD fragment 140R / 141A and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCGCACCGCGTGGG-3'; MifS-SD fragments 141A / 142E and the downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGGCGCGCACCGCGT-3'; MifS-SD fragment 142E / 143D and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTCTCGGCGCGCACCG-3'; MifS-SD fragment 143D / 144G and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCCTCGGCGCGCACC-3'; MifS-SD fragment 144G / 145S and partially linearized plasmid backbone downstream primers: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCCGTCCTCGGCGCGC-3'; MifS-SD fragment 145S / 146F and partially linearized plasmid backbone downstream primers: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCTGCCGTCCTCGGCG-3'; MifS-SD fragment 211R / 212Q and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGCGATCGTATTGCCG-3'; MifS-SD fragment 212Q / 213P and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTCTGGCGATCGTATTG-3'; MifS-SD fragment 213P / 214L and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGGGCTGGCGATCGT-3'; MifS-SD fragment 214L / 215T and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTCAGGGGCTGGCGATC-3'; MifS-SD fragment 215T / 216P and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTTGTCAGGGGCTGGCG-3'; MifS-SD fragment 216P / 217L and the downstream primer of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTGGGTGTCAGGGGC-3'; MifS-SD fragment 217L / 218R and the downstream primer of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTCAGGGGTGTCAGGGG-3'; MifS-SD fragment 218R / 219H and the downstream primer of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTGCGCAGGGGTGTC-3'; MifS-SD fragment 224S / 225Y and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCTGCGCAAGGTCTGGT-3'; MifS-SD fragment 225Y / 226G and partially linearized plasmid backbone downstream primer: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTGTAGCTGCGCAAGGTCT-3'; MifS-SD fragment 226G / 227E and the downstream primer of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCCGTAGCTGCGCAAGG-3'; MifS-SD fragment 227E / 228D and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTTTCGCCGTAGCTGCGCAA-3'; MifS-SD fragment 228D / 229R and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCTTCGCCGTAGCTG-3' MifS-SD fragment 229R / 230R and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCGGTCTTCGCCGTAGCT-3' MifS-SD fragment 230R / 231L and downstream primers of the partially linearized plasmid backbone: 5'-GACGTTGTCGCTGTTGAAGCCTGCGCTACGGCGGTCTTCG-3' MifS-SD fragment 249D / 250L and downstream primers of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGTCCAGCGATTGCCA-3' MifS-SD fragment 250L / 251P and downstream primers for the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTCAGGTCCAGCGAT-3' MifS-SD fragment 251P / 252N and downstream primers of the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTGGGCAGGTCCAGCGA-3' MifS-SD fragment 252N / 253D and downstream primers for the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGTTGGGCAGGTCCAG-3' MifS-SD fragment 253D / 254G and downstream primers of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGTCGTTGGGCAGGTC-3' MifS-SD fragment 254G / 255W and downstream primers for the partially linearized plasmid backbone: 5'-CGTTGTCGCTGTTGAAGCCTGCGCTGCCGTCGTTGGGCAGG-3' MifS-SD fragment 261R / 262D and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCGCAGCAAGTGGAGGG-3' MifS-SD fragment 262D / 263T and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCGCGCAGCAAGTGG-3' MifS-SD fragment 263T / 264A and downstream primers of the partially linearized plasmid backbone: 5'-TTGTCGCTGTTGAAGCCTGCGCTGGTGTCGCGCAGCAA-3' MifS-SD fragments 264A / 265S and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTCGCGGTGTCGCGCAGCA-3' MifS-SD fragment 265S / 266I and downstream primers for the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGCTCGCGGTGTCGCGC-3' MifS-SD fragment 266I / 267Q and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGATGCTCGCGGTGTCG-3' MifS-SD fragment 267Q / 268D and downstream primers of the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTCTGGATGCTCGCGGT-3' MifS-SD fragments 268D / 269D and downstream primers for the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCCTGGATGCTCGCG-3' MifS-SD fragment 269D / 270V and downstream primers for the partially linearized plasmid backbone: 5'-GTTGTCGCTGTTGAAGCCTGCGCTGTCGTCCTGGATGCTC-3' The primers for PCR amplification of the sfcpYFP fragment are designed as follows: upstream primer for sfcpYFP: 5'-AGCGCAGGCTTCAACAGCGACAAC-3'; Downstream primer for sfcpYFP: 5'-ACAGCCACCGTTGTACTCCAGCTTG-3'.

[0104] PCR amplification Pseudomonas aeruginosa The primer design for the PAO1-derived MifS-SD fragment and partially linearized plasmid backbone is as follows: MifS-SD fragment 41A / 42L and upstream primers for the partially linearized plasmid backbone: 5'-CAAGCTGGAGTACAACGGTGGCTGTCTGGATCCGGACCTG-3'; MifS-SD fragment 42L / 43D and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGATCCGGACCTGCGCG-3'; MifS-SD fragment 43D / 44P and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCCGGACCTGCGCGCG-3'; MifS-SD fragment 51A / 52G and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGGGCCGATCGACGGCGA-3'; MifS-SD fragment 52G / 53P and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTCCGATCGACGGCGAG-3'; MifS-SD fragment 53P / 54I and upstream primer of partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTATCGACGGCGAGTT-3'; MifS-SD fragment 54I / 55D and upstream primers of the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGACGGCGAGTTGCAG-3'; MifS-SD fragment 55D / 56G and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGGCGAGTTGCAGCA-3'; MifS-SD fragment 140R / 141A and upstream primer of partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGCCGAGGACG-3'; MifS-SD fragment 141A / 142E and upstream primer of partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGAGGACGGCAGCTTCC-3'; MifS-SD fragment 142E / 143D and upstream primer of partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGACGGCAGCTTCC-3'; MifS-SD fragment 143D / 144G and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGGCAGCTTCCTGGGCG-3'; MifS-SD fragment 144G / 145S and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTAGCTTCCTGGGCG-3'; MifS-SD lower fragment 145S / 146F and upstream primers of the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTTTCCTGGGCGCAATCG-3'; MifS-SD fragment 211R / 212Q and upstream primer of partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCAGCCCCTGACAC-3'; MifS-SD fragment 212Q / 213P and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCCCCTGACACCCC-3'; MifS-SD fragment 213P / 214L and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCTGACACCCCTGCG-3'; MifS-SD fragment 214L / 215T and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTACACCCCTGCGCC-3'; MifS-SD fragment 215T / 216P and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCCCCTGCGCCACCAG-3'; MifS-SD fragment 216P / 217L and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCTGCGCCACCAGAC-3'; MifS-SD fragment 217L / 218R and upstream primers of the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCGCCACCAGACCTT-3'; MifS-SD fragment 218R / 219H and upstream primer of partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCACCAGACCTTGCG-3'; MifS-SD fragment 224S / 225Y and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTTACGGCGAAGACCGC-3'; MifS-SD fragment 225Y / 226G and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGGCGAAGACCGCCG-3'; MifS-SD fragment 226G / 227E and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGAAGACCGCCGTC-3'; MifS-SD fragment 227E / 228D and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGACCGCCGTCTGG-3'; MifS-SD fragment 228D / 229R and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTCGCCGTCTGGCGC-3' MifS-SD fragment 229R / 230R and upstream primer of the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTCGTCTGGCGCGCGGGT-3' MifS-SD fragment 230R / 231L and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTCTGGCGCGGGTGGA-3' MifS-SD fragment 249D / 250L and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCTGCCCAACGACG-3' MifS-SD fragment 250L / 251P and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTCCCAACGACGGCTG-3' MifS-SD fragment 251P / 252N and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTAACGACGGCTGGAC-3' MifS-SD fragment 252N / 253D and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGACGGCTGGACCC-3' MifS-SD fragment 253D / 254G and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGGCTGGACCCTC-3' MifS-SD fragment 254G / 255W and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTTGGACCCTCCACTT-3' MifS-SD fragment 261R / 262D and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGACACCGCGAGCA-3' MifS-SD fragment 262D / 263T and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTACCGCGAGCATCCAGG-3' MifS-SD fragment 263T / 264A and upstream primers for the partially linearized plasmid backbone: 5'-AAGCTGGAGTACAACGGTGGCTGTGCGAGCATCCAGGA-3' MifS-SD fragment 264A / 265S and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTAGCATCCAGGACGA-3' MifS-SD fragment 265S / 266I and upstream primer of the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTATCCAGGACGACG-3' MifS-SD fragment 266I / 267Q and upstream primer of the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTCAGGACGACGTCT-3' MifS-SD fragment 267Q / 268D and upstream primers for the partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGTGACGACGTCTAGA-3' MifS-SD fragment 268D / 269D and upstream primer of partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGACGTCTAGAAG-3' MifS-SD fragment 269D / 270V and upstream primer of partially linearized plasmid backbone: 5'-CACAAGCTGGAGTACAACGGTGGCTGTGTCTAGAAGCT-3' (3) Optimization of 2-KG biosensor Further with FiKG 0.1 Based on this, targeting FiKG 0.1 A random mutant library was constructed using linkers connecting sfcpYFP and MifS-SD, and combined with high-throughput screening technology mediated by a fluorescence microplate reader to achieve FiKG... 0.1 Optimization of response amplitude. Using recombinant plasmid pET28a-FiKG 0.1 (i.e. pET28a-MifS-SD) 29-215 -sfcpYFP-MifS-SD 216-298 Using ) as a template, the sfcpYFP fragment containing a random linker was amplified by PCR using degenerate primers (ON-mutation and OC-mutation); and the recombinant plasmid pET28a-FiKG was used. 0.1 Using a template, reverse PCR amplification yielded pET28a-FiKG fragments broken at 215T and 216P. 0.1 Linearized plasmid backbone; the sfcpYFP fragment containing random linkers and the linearized plasmid backbone were recombined using a DNA assembly method based on T5 exonuclease to obtain a recombinant plasmid encoding a sensor variant containing random linkers. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and plated on LB solid plates containing kanamycin sulfate to screen for successfully constructed strains. The linker random mutant library was screened according to the screening steps in (1) above, and random mutants with high response amplitude were screened and sequenced to obtain the sensor sequence ( Figure 4 Among the 672 randomly selected mutants, the sensor corresponding to the N-terminal linker mutation of sfcpYFP to "alanine" and the C-terminal linker mutation to "leucine-proline" exhibited the highest response amplitude to 2-KG. Analysis of its dose-response curve for 2-KG showed that this sensor variant responded to 2-KG with a Δ...R max Increased to 1620.24 ± 24.36%, K d The value was 194.79 ± 12.18 μM ( Figure 5 It was named FiKG and used to establish methods for intracellular detection and enzyme activity detection of 2-KG.

[0105] FiKG containing random linkers was obtained by PCR amplification. 0.1 The variant primer design is as follows: 1N-mutation: 5'-CGCCAGCCCCTGACANNBTTCAACAGCGACAA-3' 2N-mutation: 5'-CGCCAGCCCCTGACANNBNNBTTCAACAGCGACAA-3' 3N-mutation: 5'-CGCCAGCCCCTGACANNBNNNNBTTTCAACAGCGACAA-3' 1C-mutation: 5'-TCTGGTGGCGCAGGGGVNNGTTTGTACTCCAGCT-3' 2C-mutation: 5'-TCTGGTGGCGCAGGGGVNNVNNGTTGTACTCCAGCT-3' 3C-mutation: 5'-TCTGGTGGCGCAGGGGVNNVNNVNNGTTGTACTCCAGCT-3' Reverse PCR upstream primer: 5'-CCTGCGCCACCAGACCTTGC-3' Reverse PCR downstream primer: 5'-AGGGGCTGGCGATCGTATTGC-3' Example 2: Application of 2-KG biosensor in real-time detection of 2-KG in live bacteria (1) Functional identification of FiKG in Escherichia coli W3110 The plasmid pET28a-FiKG constructed in Example 1 was transformed into... E. coli In W3110, the expression strain for FiKG was constructed. E. coli W3110-pET28a-FiKG. This strain was inoculated at a 1% inoculum in 50 mL LB medium and cultured at 37°C and 180 rpm until OD500. 600Approximately 0.6-0.8, add 1 mM IPTG, and induce FiKG expression overnight at 23℃ and 160 rpm. Collect bacterial cells by centrifugation at 6,000 rpm and 4℃ for 10 minutes, wash twice with Tris-HCl buffer, and then resuspend in Tris-HCl buffer supplemented with 9.5 mM EDTA, 0.1% toluene, and 1% glycerol to OD. 600 5. After mixing, let stand for 4 hours to induce bacterial permeation and excretion of endogenous 2-KG. Dilute the permeated bacterial solution 10-fold with Tris-HCl buffer. Take 99 μL of the bacterial solution and mix it with 1 μL of a gradient concentration of 2-KG or glucose, L-glutamate, L-glutamine, citric acid, and isocitrate in a black 96-well plate. Continuously read the fluorescence intensity using a PerkinElmer Ensight fluorescence microplate reader. Instrument parameters: excitation wavelengths of 405 nm and 488 nm, emission wavelengths of 528 nm, temperature of 37 ℃, rotation speed of 180 rpm, and detection interval of 1 minute. Results are as follows: Figure 6 As shown, in E. coli The FiKG expressed in W3110 can respond in a dose-dependent manner to the addition of gradient concentrations of exogenous 2-KG, and has high specificity for 2-KG. It does not show significant changes in fluorescence ratios for glucose, L-glutamate, L-glutamine, citric acid, and isocitrate.

[0106] (2) Application of FiKG in the detection of 2-KG in Escherichia coli W3110 exist E. coli In this process, the 2-KG permease (KgtP) transports 2-KG into the cell. To determine whether FiKG is responsive... E. coli Endogenous 2-KG fluctuations, in E. coli W3110 and E. coli W3110- kgtP The FiKG expression is used. For example... Figure 7 As shown in Figure A, FiKG is expressed as... E. coli The W3110 responds in real time to the addition of 2-KG of external energy, and under the same conditions... E. coli W3110- kgtP The fluorescence ratio remained unchanged. E. coli In this process, 2-KG can be synthesized by isocitrate dehydrogenase (ICDH) and d-2-hydroxyglutarate dehydrogenase (YdiJ); and broken down by 2-KG dehydrogenase and phosphoglycerate dehydrogenase (SerA). E. coli W3110- icdA , E. coli W3110- ydiJ , E. coli W3110- sucA , E. coli W3110- serA and E. coli W3110- serA ydiJ FiKG was expressed in the cells, and FiKG could detect the accumulation of endogenous 2-KG caused by the knockout of 2-KG dehydrogenase and phosphoglycerate dehydrogenase, as well as the decrease in intracellular 2-KG levels caused by the knockout of isocitrate dehydrogenase and d-2-hydroxyglutarate dehydrogenase. Figure 7 B).

[0107] Example 3: Application of FiKG in real-time monitoring of intracellular 2-KG in mammalian cells (1) Functional identification of FiKG in HEK293FT human embryonic kidney cells The FiKG nucleic acid sequence was optimized for mammalian codons and synthesized in its entirety by General Biosystems (Anhui) Co., Ltd. A kozark sequence (5'-GCCACC-3') was added before the start codon and ligated into pcDNA3.1. (+) plasmids, and stored in Escherichia coli Top 10 strains. Recombinant plasmid pcDNA3.11 was extracted from this strain. (+) FiKG was transfected into HEK293FT cells. Twenty-four hours after transfection, cells were washed twice with 1× Hank's balanced salt buffer containing 20 mM HEPES. Cells were then subjected to fluorescence imaging at 30-second intervals under a Zeiss 900 laser confocal microscope. Instrument parameters were set as follows: excitation wavelengths of 405 nm and 488 nm, and emission wavelengths from 497 nm to 617 nm. Cells were first treated with 80 μM digitalis saponin in the imaging buffer to infiltrate them. After 5 minutes, 5 mM 2-KG was added, and the response of FiKG expressed in HEK293FT cells to 2-KG was continuously imaged. Figure 8 As shown, FiKG responds rapidly and in real time to the addition of 2-KG, demonstrating its applicability for real-time detection of 2-KG in mammalian cells.

[0108] (2) Application of FiKG in 2-KG detection in different subcellular compartments To analyze the metabolic pathways of 2-KG in different subcellular compartments, nuclear exit sequences, mitochondrial localization sequences, and nuclear localization sequences were fused to the N-terminus, N-terminus, and C-terminus of FiKG, respectively. Figure 9As shown in Figure A, compared to FiKG, FiKG fused with different localization sequences successfully localized to the cytoplasm, mitochondria, and nucleus. Further, as described above, exogenous addition of 5 mM 2-KG to HEK293FT cells expressing FiKG with different subcellular compartment localization sequences was performed, and the change in the fluorescence ratio of FiKG before and after 2-KG addition was imaged using laser confocal microscopy. Figure 9 As shown in B, FiKG located in the cytoplasm, mitochondria, and nucleus can all respond to the addition of exogenous 5 mM 2-KG with the same response amplitude, proving that it can be used for in situ detection of 2-KG in mammalian cells.

[0109] Example 4: Application of FiKG in establishing an enzyme activity detection method The culture medium used in this embodiment: AB Inorganic Salt Medium: 10% Solution A, 1% Solution B, 1 g / L NH4Cl, adjust pH to 7.0.

[0110] Liquid A: 22.6 g / L KH2PO4, 53.7 g / L K2HPO4 3H2O, 25.3 g / L NaH2PO4 2H₂O, 84.2 g / L Na₂HPO₄ 12H₂O. Solution B: 14.8 g / L MgSO₄ 7H2O, 0.55 g / L FeSO4 7H2O, 0.045 g / L MnSO4 4H2O, 2 drops of concentrated sulfuric acid.

[0111] Exogenous expression and isolation purification were derived from Streptomyces ( Streptomyces L-glutamate oxidase (L-GOX) of sp.) X-119-6, Corynebacterium glutamicum (sp.) Corynebacterium glutamicum ATCC 13032 aspartate aminotransferase (AspB) and Bacillus subtilis ( Bacillus subtilis K2I17 isoleucine dioxygenase IDO.

[0112] (1) Detection of L-glutamate oxidase activity by FiKG L-glutamate oxidase (L-GOX) catalyzes the production of 2-KG from L-glutamate. Purified FiKG and L-glutamate oxidase were diluted to appropriate concentrations using Tris-HCl buffer. 200 μM L-glutamate and 1 μM FiKG were mixed, and 90 μL of the mixture was transferred to a black 96-well plate. 10 μL of L-glutamate oxidase was added, and the mixture was immediately mixed and used to continuously record the fluorescence intensity of FiKG using a PerkinElmer Ensight microplate reader. Instrument settings: excitation wavelengths of 405 nm and 488 nm, and emission wavelengths of 528 nm. The ratio of the fluorescence intensity measured at 488 nm excitation to that measured at 405 nm excitation (F0) was calculated. 488 nm / F 405 nm Substituting these values ​​into the measured dose-response curve of FiKG to 2-KG, the specific concentration of 2-KG can be obtained. Figure 10 A). Based on the change in 2-KG concentration and enzyme concentration per unit time, the activity of the enzyme can be calculated. The measured enzyme activity is basically consistent with the enzyme activity measured by the horseradish peroxidase (HRP) coupling method. Figure 10 B).

[0113] (2) Detection of aspartate aminotransferase activity by FiKG Aspartate aminotransferase (AspB) catalyzes the reaction of aspartate and 2-KG to oxaloacetate and glutamate. Purified FiKG and aspartate aminotransferase were diluted to appropriate concentrations using Tris-HCl buffer. A mixture of 400 μM aspartate, 200 μM 2-KG, 40 μM pyridoxal phosphate, and 1 μM FiKG was incubated for 10 minutes. 90 μL of the mixture was then transferred to a black 96-well plate, and 10 μL of aspartate aminotransferase was added. After mixing, the fluorescence intensity of FiKG was immediately recorded continuously using a PerkinElmer Ensight microplate reader. Figure 11 A). Instrument parameter settings and data processing are as described above. The results of aspartate aminotransferase activity measured by FiKG and malate dehydrogenase (MDH) coupling method are basically consistent. Figure 11 B).

[0114] (3) Detection of isoleucine dioxygenase activity by FiKG Isoleucine dioxygenase (IDO) is a 2-oxoglutaric acid-dependent dioxygenase that catalyzes the hydroxylation of isoleucine to produce (2... S ,3 R 4 S4-Hydroxyisoleucine. This product possesses multiple physiological functions, including promoting insulin secretion, improving insulin resistance, and regulating blood lipids, and is considered one of the most promising oral antidiabetic drug candidates. Therefore, establishing a rapid and sensitive enzyme activity detection method and developing an efficient high-performance mutant screening platform are crucial for promoting the application of this enzyme and accelerating the development of related drugs. The purified FiKG and isoleucine dioxygenase were diluted to appropriate concentrations using Tris-HCl buffer. 1 mM l-isoleucine, 200 μM 2-KG, 50 μM ferrous sulfate, 2 mM L-ascorbic acid, and 1 μM FiKG were mixed and incubated for 10 minutes. 90 μL of the mixture was then transferred to a black 96-well plate, and 10 μL of isoleucine dioxygenase was added. After mixing, the fluorescence intensity of FiKG was immediately recorded continuously using a PerkinElmer Ensight fluorescence microplate reader. Figure 12 A). Instrument parameter settings and data processing are as described above. Isoleucine dioxygenase activity and (2) were measured based on FiKG. S ,3 R 4 S The results obtained by the 4-hydroxyisoleucine dehydrogenase coupling method were basically consistent. Figure 12 B).

[0115] (4) Analysis of the application potential of FiKG in the screening of high-performance mutants of 2-KG-related enzymes The plasmid pACYC-Duet-IDO carrying wild-type isoleucine dioxygenase and the isoleucine dioxygenase mutant I162T / T182N (a high-performance IDO mutant reported in the literature) were used to... I162T / T182N The strains were transferred into *E. coli* containing pET28a-FiKG. The above strains were inoculated at a 1% inoculum in 50 mL of LB medium and cultured at 37°C and 180 rpm until OD500. 600 Approximately 0.6-0.8 mg / L, add 1 mM IPTG, and incubate overnight at 23°C and 160 rpm to induce FiKG and isoleucine dioxygenase expression. Collect an appropriate amount of bacterial culture, centrifuge at 6,000 rpm for 10 minutes to collect the cells, wash twice with AB inorganic salt medium, and resuspend in AB inorganic salt medium to OD200. 600 5. The resuspension was transferred to a 96-well plate, and the fluorescence intensity of FiKG was detected using a PerkinElmer Ensight fluorescence microplate reader. The instrument parameters and data processing were as described above, thus obtaining the differences in 2-KG levels among strains expressing different isoleucine dioxygenases. Figure 13 This indicates that FiKG can be used to screen for high-performance mutants of 2-KG-related enzymes.

[0116] Example 5: Application of FiKG in high-throughput screening of enzyme inhibitors Taking the screening of inhibitors of 2-oxoglutarate-dependent dioxygenase-proline hydroxylase 2 as an example, this paper illustrates the operation process of the high-throughput screening method for enzyme inhibitors based on the 2-KG biosensor FiKG.

[0117] Hypoxia-inducible factor-1 (HIF-1) is a hypoxia-associated transcription factor, a heterodimer composed of α and β subunits, that regulates gene expression involved in processes such as angiogenesis and erythropoiesis. Under normoxic conditions, proline hydroxylase (PHD) catalyzes the hydroxylation of prolines at positions 402 and 564 of HIF-1α. PHDs are a class of oxygen-dependent, 2-KG, and ferrous ion-catalyzed dioxygenases. PHD2 is one of the most widely distributed PHD isoforms and is frequently used for screening PHD inhibitors.

[0118] The hypoxia-inducible factor-1α short peptide (HIF-1αC19) in this embodiment was synthesized by Shanghai Hongtai Biotechnology Co., Ltd. An FDA-approved drug library (purchased from MedChemExpress) was used as the inhibitor screening library. Exogenous expression, isolation, and purification were performed on human proline hydroxylase 2 (PHD2). 181-426 ).

[0119] The PHD2 inhibitor screening method in this embodiment includes mixing 200 μM 2-KG, 50 μM ferrous sulfate, 2 mM L-ascorbic acid, 1 μM FiKG, and 40 μM of different compounds, adding 90 μL of the mixture to a black 96-well plate, incubating at room temperature for 10 minutes, and then adding 10 μL of PHD2. 181-426 After mixing, the fluorescence intensity of FiKG was immediately recorded continuously using a PerkinElmer Ensight fluorescence microplate reader. Instrument parameters were set as described in Example 4. The ratio of the fluorescence intensity measured at 488 nm excitation to that measured at 405 nm excitation (FKG) was calculated. 488 nm / F 405 nm The rate of decrease in fluorescence ratio per unit time can be obtained by measuring the decrease in fluorescence ratio. Under the same conditions, the change in fluorescence intensity of the reaction system with added DMSO was measured by FiKG as a control. Compared with the control group, the lower the rate of decrease in fluorescence ratio, the weaker the PHD2 activity and the higher the inhibition efficiency of the compound.

[0120] This embodiment screened 2906 compounds from the FDA-approved drug library, among which 42 compounds showed inhibitory activity as high as 90%. These 42 compounds then underwent a second round of screening, using the methods described above. Figure 14As shown, four compounds—troglitazone, dipyridamole, Evans Blue, and vadadustat—almost completely inhibited PHD2 activity. Among them, vadadustat is a marketed PHD2 inhibitor, troglitazone is a hypoglycemic drug, dipyridamole is an anti-tumor drug, and no drug activity related to Evans Blue has been reported.

[0121] In this embodiment, FiKG was used to determine the half-maximal inhibitory concentration (IC50) of Evans blue and vardadustrol against PHD2 in vitro. Figure 15 The detection method involves mixing 200 μM 2-KG, 50 μM ferrous sulfate, 2 mM L-ascorbic acid, 1 μM FiKG, and gradient concentrations of Evans blue or vardadustat. 90 μL of this mixture is added to a black 96-well plate and incubated at room temperature for 10 minutes. Then, 10 μL of PHD2 is added. 181-426 After mixing, the fluorescence intensity of FiKG was immediately recorded continuously using a PerkinElmer Ensight fluorescence microplate reader. Instrument parameter settings and data processing were as described in Example 4.

[0122] The nucleotide / amino acid sequences involved in this invention FiKG amino acid sequence: RQARQQALRAEGEQVRKQLDLYAGSLQTLIERFRSLPAVLALDPDLRAALAGPIDGELQQRLNLKLESINLAARSSTLELLDRTGLAVAASNWNLPTSYVGHNYGFRPYFRQTIAQGSGRFYAVGVISGIPGYFLSHAVRAEDGSFLGAIVVKLEFPDLERQWNQTPDLVLASDAKGIVFLANHAGWRYRELEPLDTVDRFELAETRQYDRQPLTAFNSDNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSFQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYNVDGGSGGTGSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKLICTTGKLPVPWPTLVTTLGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIGFKEDGNILGHKLEYNLPPLRHQTLRSYGEDRRLARVESADGEKDYLWQSLDLPNDGWTLHLLRDTASIQDDV (SEQ ID NO.1) Nucleic acid sequence of FiKG: Matters not covered in this invention are common knowledge.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein includes the MifS extracellular sensing domain and a fluorescent protein; wherein the extracellular sensing domain of MifS includes, but is not limited to, the extracellular sensing domain of MifS from Pseudomonas aeruginosa (PAO1) Pseudomonas aeruginosa ) MifS of the MifS / MifR two-component signal transduction system from PAOl The fluorescent protein is specifically a cyclic rearranged fluorescent protein, further including any one or more of cyclic rearranged cyan fluorescent protein, cyclic rearranged green fluorescent protein, cyclic rearranged yellow fluorescent protein, and cyclic rearranged red fluorescent protein; Furthermore, the fusion protein is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1; (a2) A protein having the same or similar function by substituting, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in (a1). (a3) has an amino acid sequence composition that is 40% or more identical to that shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 1; Furthermore, the nucleic acid molecule has any of the nucleotide sequences described in (b1)–(b4): (b1) The nucleotide sequence as shown in SEQ ID NO.2; (b2) A sequence formed by substitution, deletion and / or addition of one or more nucleotides as shown in (b1); (b3) has 40% or more identity with the nucleotide sequence defined in (b1) or (b2) and is a nucleic acid molecule encoding the fusion protein; (b4) A nucleotide sequence that, under stringent conditions, can hybridize with any of the nucleotide sequences described in (b1)–(b3) and encode a fusion protein with the same function.

3. A recombinant expression vector, characterized in that, The recombinant expression comprises at least the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2, the recombinant expression vector of claim 3, or is capable of expressing the fusion protein of claim 1.

5. The use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3, and / or the host cell of claim 4 in the preparation of a biosensor for detecting 2-KG.

6. A biosensor for detecting 2-KG, characterized by, The biosensor comprises at least the fusion protein as described in claim 1; Furthermore, the biosensor also includes other reagents, devices, and / or equipment for 2-KG detection.

7. A method for detecting 2-KG in vitro, characterized in that, The method includes at least: incubating the sample to be tested with the fusion protein of claim 1 or the biosensor of claim 6, and analyzing the concentration or presence of 2-KG in the sample to be tested based on the change in fluorescence signal of the biosensor; The sample to be tested is any biological or environmental sample containing 2-KG or suspected of containing 2-KG.

8. A method for intracellular detection of 2-KG, characterized in that, The method includes at least: inducing the expression of the fusion protein of claim 1 or the biosensor of claim 6 in cells, and analyzing the concentration or presence of 2-KG in cells based on changes in the fluorescence signal of the biosensor; Furthermore, the cells are bacterial cells, fungal cells, or animal cells.

9. A method for detecting the activity of 2-KG-related enzymes or for high-throughput screening of 2-KG-related enzyme materials, characterized in that, The method includes: contacting the test sample containing or suspected of containing 2-KG-related enzymes with the fusion protein of claim 1 or the biosensor of claim 6, and analyzing the activity of the 2-KG-related enzymes to be tested based on the changes in the fluorescence signal of the biosensor; Furthermore, the 2-KG-related enzymes include, but are not limited to, L-glutamate oxidase, aspartate aminotransferase, isoleucine dioxygenase, and human proline hydroxylase 2 and its mutants. Still further, the 2-KG related enzyme is human prolyl hydroxylase 2 (PHD2 181-426 ) The 2-KG-related enzyme material includes 2-KG-related enzyme inhibitors or 2-KG-related enzyme promoters.

10. The use of the fusion protein of claim 1, the biosensor of claim 6, and / or the method of any one of claims 7-9 in the detection of 2-KG-related diseases and / or the screening of related drugs.

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