CN116949001A - An L-2-hydroxyglutarate biosensor based on highly catalytically active L-2-hydroxyglutarate dehydrogenase and its application - Google Patents

Abstract

The invention belongs to the field of biological detection technology, and specifically relates to an L-2-hydroxyglutarate biosensor based on highly catalytically active L-2-hydroxyglutarate dehydrogenase and its application. The present invention screens out L-2-HG dehydrogenase with high catalytic activity from a variety of bacterial strains, and constructs an L-2-HG biosensor based on the high catalytic activity L-2-HG dehydrogenase. After experimental verification, The response amplitude reaches 2189.25±26.89%, and the detection limit is as low as 0.042μM, which enables quantitative detection of L‑2‑HG in a variety of bacteria, cell samples and human body fluids, and can be applied to basic research related to L‑2‑HG. , detection of L‑2‑HG related diseases (such as kidney cancer) and screening of L‑2‑HG related drugs (such as L‑2‑HG activators or inhibitors), etc., so it has broad application prospects.

Description

A kind of L-2-hydroxyglutarate based on highly catalytically active L-2-hydroxyglutarate dehydrogenase Acid biosensors and their applications

Technical field

The invention belongs to the field of biological detection technology, and specifically relates to an L-2-hydroxyglutarate biosensor based on highly catalytically active L-2-hydroxyglutarate dehydrogenase and its application.

Background technique

The information in this Background section is disclosed solely for the purpose of increasing understanding of the general background of the invention and is not necessarily considered to be an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

L-2-Hydroxyglutarate (L-2-HG) is a structural analog of 2-ketoglutarate (2-KG) in the TCA cycle. Previous studies have shown that L-2-HG plays diverse biological functions in the normal physiological metabolism of living organisms, including participating in L-lysine decomposition and carbon starvation response, hypoxia adaptation, early embryonic development and immune response. In mammals and microorganisms, the decomposition of L-2-HG depends on the flavoprotein L-2-HG dehydrogenase (called L2HGDH in mammals and LhgO in microorganisms) with FAD as a cofactor. Convert it to 2-KG. L2HGDH mutations can lead to abnormal accumulation of L-2-HG, which inhibits the activity of multiple 2-KG-dependent dioxygenases, leading to genome-wide changes in histone and DNA methylation, preventing cell differentiation, and ultimately causing cancer. occurs, and the accumulation of L-2-HG has been found in various diseases such as L-2-hydroxyglutaric aciduria, renal cancer, pancreatic cancer, and colorectal cancer.

L-2-HG is a key signaling and immune metabolite in living organisms, and it can be used as a key biomarker for the diagnosis and prognosis of L-2-HG-related diseases. It is accurate, convenient, high-throughput and low-cost to develop. The L-2-HG detection method has important scientific value and clinical significance. The current detection of L-2-HG mainly relies on technical methods such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), and magnetic resonance spectroscopy (MRS). These methods All are expensive, cumbersome, time-consuming, difficult to separate L-2-HG and its enantiomer D-2-hydroxyglutarate (D-2-HG), and require professional experiments. Operators are required, and it is incompatible with high-throughput analysis, which limits the development of L-2-HG-related disease diagnosis and treatment and functional diversity research. In addition, a L-2-HG biosensor LHGFR based on L-2-HG-specific transcriptional regulators and fluorescence resonance energy transfer technology was recently reported. After optimization, the maximum fluorescence signal changes (ΔR _max , that is, the response amplitude) of the sensors LHGFR _0N3C and LHGFR _0N7C to L-2-HG were 56.13±0.29% and 60.37±1.30%, respectively. In addition, the lower detection limits (LOD) of LHGFR _0N3C and LHGFR _0N7C for L-2-HG in serum are 5.84 μM and 1.68 μM, respectively, and the lower detection limits of L-2-HG in urine are 15.74 μM and 0.92 μM, respectively. The lower response amplitude and higher detection limit limit LHGFR in accurate quantification of low-concentration L-2-HG, such as the inability to detect L-2-HG in body fluids of healthy adults. Therefore, there is an urgent need to develop a high-performance L-2-HG biosensor with high throughput, low cost, high response amplitude, and low detection limit to meet the needs of quantitative detection of L-2-HG.

Contents of the invention

In view of the shortcomings of the above-mentioned existing technologies, the inventor has provided an L-2-HG biosensor based on highly catalytic activity L-2-HG dehydrogenase and its application after long-term technical and practical exploration. The present invention screens out L-2-HG dehydrogenase with high catalytic activity from a variety of strains, and constructs and obtains an L-2-HG biosensor based on the high catalytic activity L-2-HG dehydrogenase. After experimental verification, the L-2-HG biosensor has a response amplitude of 2189.25±26.89% and a detection limit as low as 0.042 μM, thereby enabling the quantification of L-2-HG in a variety of bacteria, cell samples, and human body fluids. detection. Based on the above research results, the present invention is completed.

In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a high catalytic activity L-2-HG dehydrogenase, the high catalytic activity L-2-HG dehydrogenase is derived from bacteria including but not limited to Azoarcus olearius BH72, Any one or more of Pandoraeasputorum NCTC13161 and Indibacter alkaliphilus LW1.

Specifically, the L-2-HG dehydrogenase is selected from:

(a1) A protein consisting of the amino acid sequence shown in any one of SEQ ID NO. 1-3;

(a2) A protein that has the same or similar functions by substituting, deleting and/or adding one or more amino acid residues to the amino acid sequence shown in (a1);

(a3) A protein that has an identity of 50% or more with the amino acid sequence composition shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

A second aspect of the present invention provides a polynucleotide capable of encoding the above-mentioned high catalytic activity L-2-HG dehydrogenase.

Specifically, the polynucleotide has any one of the nucleotide sequences (b1)-(b4):

(b1) The nucleotide sequence shown in SEQ ID NO.4-6;

(b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1);

(b3) A nucleic acid molecule that has 50% or more identity with the nucleotide sequence defined in (b1) or (b2) and encodes the fusion protein;

(b4) A nucleotide sequence capable of hybridizing to the nucleotide sequence described in any one of (b1) to (b3) under stringent conditions and encoding a highly catalytically active L-2-HG dehydrogenase with the same function.

A third aspect of the present invention provides a recombinant expression vector, which at least contains the above polynucleotide.

A fourth aspect of the present invention provides a host cell, which contains the above-mentioned polynucleotide, the above-mentioned recombinant expression vector or is capable of expressing the above-mentioned high catalytic activity L-2-HG dehydrogenase.

The fifth aspect of the present invention provides the application of the above-mentioned highly catalytically active L-2-HG dehydrogenase, polynucleotide, recombinant expression vector and/or cells in preparing a biosensor for detecting L-2-HG.

The sixth aspect of the present invention provides a biosensor for detecting L-2-HG (which can be named EaLHGFR). The biosensor at least contains the above-mentioned high catalytic activity L-2-HG dehydrogenase, and an oxidizing Reduction reaction indicator; wherein, the redox reaction indicator can be an electron acceptor that can accept electrons generated by L-2-HG dehydrogenase catalyzed by L-2-HG dehydrogenase.

In actual application, the above-mentioned biosensor can exist in the form of a detection kit.

A seventh aspect of the present invention provides a method for detecting L-2-HG, which method at least includes: incubating the sample to be tested with the biosensor, and analyzing the concentration or presence of L-2-HG.

Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects:

(1) The above technical solution is based on sequence comparison analysis to screen out multiple L-2-HG dehydrogenases with high catalytic activity from different strains; among them, one with high substrate activity was identified in A.olearius BH72 AoL2HGDH, a specific and highly catalytically active L-2-HG dehydrogenase; AoL2HGDH uses FAD as a cofactor, catalyzes the conversion of L-2-HG into 2-KG and can directly transfer electrons to the redox reaction indicator without the need for Additional electron transfer mediator;

(2) The L-2-HG biosensor EaLHGFR based on the highly catalytic activity L-2-HG dehydrogenase developed by the above technical solution has the characteristics of high response amplitude and low detection limit, and the detection system only contains AoL2HGDH and redox The two-component reaction indicator is easy to prepare, simple in composition, low in cost, easy to operate, and can achieve high-throughput detection;

(3) The L-2-HG biosensor EaLHGFR based on the highly catalytic activity L-2-HG dehydrogenase developed by the above technical solution is suitable for L-2- in different bacteria, cells, human serum, urine and other biological samples. The quantitative detection of HG, the detection results are highly consistent with the current L-2-HG standard detection method LC-MS/MS, and it has broad application prospects in the detection of L-2-HG in various biological samples.

Description of the drawings

The description and drawings that constitute a part of the present invention are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Figure 1 is a comparison of the enzyme activities of L-2-HG dehydrogenases from different sources in Example 1 of the present invention.

Figure 2 is SDS-PAGE verification of expression and purification of AoL2HGDH in Example 1 of the present invention.

Figure 3 is the measurement of kinetic parameters and substrate specificity analysis of AoL2HGDH on L-2-HG in Example 1 of the present invention; A is the measurement of kinetic parameters of AoL2HGDH on L-2-HG, and B is the substrate of AoL2HGDH. Substance-specific analysis.

Figure 4 is a schematic diagram of the detection principle of the L-2-HG biosensor based on AoL2HGDH in Embodiment 2 of the present invention.

Figure 5 is a dose-response curve of the initial L-2-HG biosensor EaLHGFR-1 to L-2-HG in Example 2 of the present invention.

Figure 6 shows the system optimization of the L-2-HG biosensor in Example 2 of the present invention.

Figure 7 is a dose-response curve of L-2-HG for the optimized L-2-HG biosensor EaLHGFR-2 in Example 2 of the present invention.

Figure 8 shows the consistency analysis of EaLHGFR-2 and LC-MS/MS for the detection of bacterial sample L-2-HG in Example 3 of the present invention; wherein, A is the use of EaLHGFR-2 and LC-MS/MS for bacterial lysis. Consistency analysis of L-2-HG detection in bacteria, B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for L-2-HG detection in bacterial culture media.

Figure 9 shows the quantitative detection of L-2-HG inside and outside bacterial cells using EaLHGFR-2 in Example 3 of the present invention; where A is the result of quantitative detection of L-2-HG inside bacterial cells, and B is the results of quantitative detection of L-2-HG outside bacterial cells. Results of quantitative detection of 2-HG.

Figure 10 is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detecting L-2-HG in cell samples in Example 4 of the present invention; wherein, A is EaLHGFR-2 and LC-MS/MS for cell lysis. Consistency analysis of L-2-HG detection in substances, B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for L-2-HG detection in cell culture media.

Figure 11 shows the quantitative detection of intracellular L-2-HG using EaLHGFR-2 in Example 4 of the present invention; where A is the result of quantitative detection of intracellular L-2-HG, and B is the result of extracellular L-2-HG. Results of quantitative detection of 2-HG.

Figure 12 is the consistency analysis of EaLHGFR-2 and LC-MS/MS for the detection of L-2-HG in human body fluids in Example 5 of the present invention; wherein, A is the use of EaLHGFR-2 and LC-MS/MS for human serum Consistency analysis of L-2-HG detection in medium, B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for L-2-HG detection in human urine.

Figure 13 shows the quantitative detection of L-2-HG in human body fluids using EaLHGFR-2 in Example 5 of the present invention; where A is the result of quantitative detection of L-2-HG in human serum, and B is L-2 in human urine. -Results of HG quantitative testing.

Detailed ways

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular forms are also intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, it will be understood that when the terms "comprises" and/or "includes" are used in this specification, they indicate There are features, steps, operations, means, components and/or combinations thereof.

In a typical embodiment of the present invention, a high catalytic activity L-2-HG dehydrogenase is provided, and the high catalytic activity L-2-HG dehydrogenase is derived from bacteria including but not limited to Azoarcus olearius. Any one or more of BH72, Pandoraea sputorum NCTC13161 and Indibacter alkaliphilus LW1; further derived from Azoarcus oearius BH72, at this time, the highly catalytic activity L-2-HG The dehydrogenase is named AoL2HGDH, and its GeneBank number is CAL94536.1; AoL2HGDH covalently binds FAD as a cofactor, catalyzes L-2-HG to generate 2-KG, and has high substrate specificity and high dehydration of L-2-HG. Hydrogen activity.

The L-2-HG dehydrogenase is selected from:

(a1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1-3;

(a2) A protein that has the same or similar functions by substituting, deleting and/or adding one or more amino acid residues to the amino acid sequence shown in (a1);

(a3) A protein that has an identity of 50% or more with the amino acid sequence composition shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2).

Wherein, in (a2), the substitution, deletion and/or addition of one or more amino acid residues is generally a substitution, deletion and/or addition of no more than 15 amino acid residues.

The proteins shown in (a1)-(a3) above can be synthesized artificially, or their encoding genes can be synthesized first and then obtained through biological expression.

In one or more specific embodiments of the present invention, a polynucleotide is provided, which can encode the above-mentioned high catalytic activity L-2-HG dehydrogenase.

Specifically, the polynucleotide has any one of the nucleotide sequences (b1)-(b4):

(b1) The nucleotide sequence shown in SEQ ID NO.4-6;

(b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1);

(b3) A nucleic acid molecule that has 50% or more identity with the nucleotide sequence defined in (b1) or (b2) and encodes the fusion protein;

(b4) A nucleotide sequence capable of hybridizing to the nucleotide sequence described in any one of (b1) to (b3) under stringent conditions and encoding a highly catalytically active L-2-HG dehydrogenase with the same function.

It should be noted that the term "identity" refers to sequence similarity to an amino acid/nucleotide sequence. Identity can be assessed with the naked eye or with 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.

The above-mentioned 50% or more identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% or more identity.

The polynucleotide may be DNA, such as cDNA, genomic DNA, or recombinant DNA, and is not specifically limited here.

In one or more specific embodiments of the present invention, a recombinant expression vector is provided, which at least contains the above polynucleotide.

The recombinant expression vector is obtained by effectively connecting the above-mentioned polynucleotide to an expression vector, and the expression vector includes viral vectors (including adenovirus vectors, retroviral vectors or adeno-associated virus vectors), plasmids, phages, mucosal vectors, particles or artificial chromosomes; further, the expression vector can be a plasmid, such as pACYCDuet-1 plasmid.

In one or more specific embodiments of the present invention, a host cell is provided, which contains the above-mentioned polynucleotide, the above-mentioned recombinant expression vector or is capable of expressing the above-mentioned high catalytic activity L-2-HG dehydrogenase.

The host cells include bacterial cells or fungal cells;

The bacteria may be any one or more of the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus.

In one or more specific embodiments of the present invention, the bacteria are Escherichia coli (such as BL21 (DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Lactococcus lactis, Bacillus subtilis, Bacillus or Pseudomonas fluorescens.

In one or more specific embodiments of the present invention, the above-mentioned high catalytic activity L-2-HG dehydrogenase, polynucleotide, recombinant expression vector and/or cells are provided in the preparation of biosensors for detecting L-2-HG. Applications.

In one or more specific embodiments of the present invention, a biosensor for detecting L-2-HG (which can be named EaLHGFR) is provided. The biosensor at least contains the above-mentioned high catalytic activity L-2-HG dehydrogenase. , and a redox reaction indicator; wherein the redox reaction indicator can be an electron acceptor that can accept electrons generated by L-2-HG dehydrogenase catalyzed by L-2-HG dehydrogenase.

The electron acceptor includes but is not limited to azure, 3'-[1-[(anilino)-carbonyl]-3,4-tetrazole]-bis(4-methoxy-6-nitro)benzene- Sodium sulfonate and dichlorophenol indophenol.

The biosensor may also include other reagents, devices and/or equipment for L-2-HG detection;

The reagents include detection buffer.

In yet another specific embodiment of the present invention, the biosensor for detecting L-2-HG includes: 0.1 to 50 μM resazurin, 0.005 to 1 mg mL ^-1 AoL2HGDH, and a buffer; the buffer is selected from Tris- HCl, HEPES, potassium phosphate, sodium phosphate, MOPS and PBS, the buffer concentration is 50-100mM, and the pH is 5.8-9.0;

Further preferably, the biosensor for detecting L-2-HG includes: 67mM PBS (pH 6.6), 1μM resazurin, 0.05mg mL ^-1 AoL2HGDH. Under this condition, when L-2-HG appears in the reaction system, AoL2HGDH catalyzes the dehydrogenation of L-2-HG, and the covalently bound FAD is converted into FADH _2. FADH ₂ further transfers electrons to resazurin, The highly red-shifted fluorescent molecule resorufin (emission peak at 587nm) is generated, and at this time, it has the highest response amplitude to 0.5μM and 1μM L-2-HG. After experimental verification, its response amplitude is 2189.25±26.89%. The lower detection limit is 0.042μM, thus achieving highly sensitive detection of L-2-HG.

In actual application, the above-mentioned biosensor can exist in the form of a detection kit.

The kit may contain materials or reagents (including highly catalytically active L-2-HG dehydrogenase, redox reaction indicators, etc.) used to implement the method of the present invention. The kit may include storage reaction reagents (eg, L-2-HG dehydrogenase, redox reaction indicator, etc. in appropriate containers) and/or supporting materials (eg, buffers, instructions for performing the assay, etc.). For example, a kit may include one or more containers (eg, boxes) containing corresponding reaction reagents and/or support materials. Such contents may be delivered together or separately to the intended recipient. For example, a first container may contain the enzyme for the assay, the second container contains the redox reaction indicator, and the third container contains the buffer. The kit may also contain compartments suitable for holding the reagents or containers. There are no specific limitations here.

In one or more specific embodiments of the present invention, a method for detecting L-2-HG is provided, which method at least includes: incubating the sample to be tested with the biosensor, analyzing the concentration of L-2-HG or Yes or no.

Wherein, the sample to be tested is a sample containing or suspected of containing L-2-HG. 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, and cell culture medium. , cell lysate, animal serum, animal urine and animal tissue fluid;

Wherein, the animal may be a mammal, among which humans are preferred.

Qualitative or quantitative detection of L-2-HG through the above methods can be applied to basic research on L-2-HG, detection of L-2-HG-related diseases (such as kidney cancer) and screening of L-2-HG-related drugs. (such as L-2-HG activator or inhibitor) and many other fields, so it has broad application prospects.

The present invention will be further described below with reference to specific examples. The following examples are only for explaining the present invention and do not limit its content. The experimental methods used are routine methods unless otherwise specified. The bacterial strains, cells, materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

Example 1: Identification and characterization of highly catalytically active L-2-HG dehydrogenase AoL2HGDH protein

(1) Comparison of exogenous expression and enzyme activities of different L-2-HG dehydrogenase homologous proteins

Based on sequence comparison analysis, the nucleotide sequences of L-2-HG dehydrogenase homologous proteins derived from ten species of bacteria, fungi and plants were obtained, synthesized by General Biosystems (Anhui) Co., Ltd. and connected to the pACYCDuet-1 plasmid. Saved in E.coli BL21 (DE3) strain; the expression strains were inserted into LB liquid culture medium containing chloramphenicol (40μg mL ^-1 ), cultured at 37°C and 180rpm until the OD _600nm was about 0.6, and 1mM was added IPTG, induce protein expression overnight at 23°C and 160 rpm; collect the cells by centrifugation, and obtain the crude enzyme solution of each L-2-HG dehydrogenase after high-pressure crushing; each L-2-HG dehydrogenase has a strong effect on L- The 2-HG activity was measured in an 800 μL reaction system, containing 50 mM Tris-HCl (pH 7.4), 100 μM DCPIP, 100 μM L-2-HG and 40 μL crude enzyme solution. Each activity was determined by detecting the change in absorbance at 600 nm at 30°C. Catalytic activity of L-2-HG dehydrogenase towards L-2-HG. The results are shown in Figure 1. Among them, the L-2-HG dehydrogenase (GeneBank: CAL94536.1) derived from A. olearius BH72 has the highest catalytic activity for L-2-HG and is named AoL2HGDH. The amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.4.

(2) Expression and purification of AoL2HGDH

After the E.coli BL21 (DE3) strain carrying the AoL2HGDH expression vector was activated for two generations in LB liquid medium, it was inoculated into 500 mL LB liquid medium containing chloramphenicol (40 μg mL ^-1 ) and incubated at 37°C and 180 rpm. Culture under conditions until OD _600nm is about 0.6, add 1mM IPTG, induce AoL2HGDH expression overnight at 23°C and 160rpm; collect the cells by centrifugation, wash twice with binding buffer and resuspend until OD _600nm is 20, add 1mM PMSF With 10% glycerol, high-pressure disrupt the cells, centrifuge at 12,000 rpm and 4°C for 50 minutes to remove cell debris and obtain the crude enzyme solution of AoL2HGDH; the crude enzyme solution is filtered through a 0.22 μm filter head and separated and purified using a 5mL nickel column. The purified AoL2HGDH was obtained by elution with the elution buffer at a certain concentration; SDS-PAGE was used to detect the purity of AoL2HGDH, and the results are shown in Figure 2.

(3) Determination of kinetic parameters and substrate specificity analysis of AoL2HGDH

The kinetic parameters of AoL2HGDH on L-2-HG were measured in an 800μL reaction system, containing 50mM Tris-HCl (pH 7.4), 100μM DCPIP, gradient concentration of L-2-HG and an appropriate amount of purified AoL2HGDH protein. Detect the change in absorbance at 600nm at ℃ to determine the catalytic activity of AoL2HGDH for gradient concentration L-2-HG, and calculate the kinetic parameters of AoL2HGDH for L-2-HG by fitting; the results are shown in Figure 3A, AoL2HGDH for L-2 The V _max value of -HG is 50.85±1.66U mg ^-1 and the K _m value is 29.12±2.52μM. The substrate specificity analysis reaction system of AoL2HGDH is the same as above, except that L-2-HG is replaced with 10 μM D-2-HG, L-tartaric acid, D-tartaric acid, L-glyceric acid, D-glyceric acid, L-lactic acid, D - For L-2-HG structural analogs such as lactic acid, the substrate specificity of AoL2HGDH was determined by measuring the activity of AoL2HGDH on different structural analogs; the results are shown in Figure 3B. AoL2HGDH has high specificity for L-2-HG.

Among them, the LB medium formula described in the above steps (1) to (2) is: yeast powder 5g L ^-1 ; peptone 10g L ^-1 ; NaCl 10g L ^-1 , pH 7.0; sterilized at 121°C for 20 minutes.

The binding buffer formula described in the above step (2) is: 20mM Na ₂ HPO ₄ , 20mM imidazole, 500mM NaCl, adjust the pH to 7.4; the elution buffer formula is: 20mM Na ₂ HPO ₄ , 500mM imidazole, 500mM NaCl , adjust the pH to 7.4.

Example 2: Construction and optimization of L-2-HG biosensor based on highly catalytically active L-2-HG dehydrogenase AoL2HGDH

(1) Construction principle of L-2-HG biosensor based on highly catalytic activity L-2-HG dehydrogenase AoL2HGDH

AoL2HGDH has high catalytic activity and high substrate specificity, and can be used as an ideal catalytic element to combine with the resazurin-mediated redox reporter system to develop an enzymatic L-2-HG biosensor (EaLHGFR). The detection principle of EaLHGFR is shown in Figure 4, in which AoL2HGDH catalyzes the dehydrogenation of L-2-HG to generate 2-KG, and at the same time reduces the covalently bound FAD to FADH ₂ ; the generated FADH ₂ can further reduce resazurin to generate high The red-shifted fluorescent molecule resorufin.

(2) Construction and characterization of the initial L-2-HG biosensor EaLHGFR-1

Prepare the L-2-HG biosensor working solution (the initial L-2-HG biosensor is named EaLHGFR-1) consisting of 50mM Tris-HCl (pH 7.4), 15μM resazurin, and 0.1mg mL ^-1 AoL2HGDH. For gradient concentration L-2-HG solution (0mM to 20mM), mix 75μL aLHGFR-1 working solution and 25μL L-2-HG solution in a black 96-well plate, react at 30°C for 60 minutes in the dark, and use luciferase The standard instrument measures the fluorescence emission intensity at 590nm under the excitation wavelength of 544nm. Taking the concentration of L-2-HG as the abscissa and the fluorescence intensity of EaLHGFR-1 as the ordinate, draw the dose-response curve of EaLHGFR-1 to L-2-HG. The results are shown in Figure 5. EaLHGFR-1 responded to the addition of L-2-HG in a dose-dependent manner. Its response amplitude (ie, maximum fluorescence intensity change, ΔF _max ) was 1933.73±38.99%, and the lower detection limit was 0.976 μM. .

(3) System optimization of L-2-HG biosensor

In order to improve the detection sensitivity of L-2-HG biosensor, a single factor screening strategy was used to target the detection buffer types (50mM Tris-HCl [pH 7.4], 100mM Tris-HCl [pH 7.4], 100mM HEPES [pH 7.4] , 100mM potassium phosphate [pH 7.4], 100mM sodium phosphate [pH 7.4], 100mM MOPS [pH 7.4] and 67mM PBS [pH 7.4]), detection buffer pH (5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2 , 8.6, 9.0), resazurin concentration (0.1, 0.5, 1, 5, 10, 15, 50μM) and AoL2HGDH concentration (0.005, 0.01, 0.05, 0.07, 0.1, 0.5, 1mg mL ^-1 ) for system optimization. The detection sensitivity of the biosensor was determined based on the response amplitude of the biosensor to low concentrations of L-2-HG (0.5 μM and 1 μM). The results are shown in Figure 6. The biosensor variant composed of 67mM PBS (pH 6.6), 1μM resazurin, and 0.05mg mL ^-1 AoL2HGDH has the highest response amplitude to 0.5μM and 1μM L-2-HG, respectively. Improved from 29% and 66% for EaLHGFR-1 to 488% and 885%, this sensor variant was named EaLHGFR-2.

(4) Characterization of the optimized L-2-HG biosensor EaLHGFR-2

The dose-response curve of EaLHGFR-2 versus L-2-HG was determined by the method described in (2). The results are shown in Figure 7. EaLHGFR-2 responded to the addition of L-2-HG in a dose-dependent manner, with a response amplitude of 2189.25±26.89% and a lower detection limit as low as 0.042 μM.

Example 3: Application of EaLHGFR-2 in quantitative detection of bacterial intracellular and extracellular L-2-HG

(1) Preparation of bacterial culture medium and bacterial lysate

Culture the P.putida KT2440 strain to the mid-log phase in an inorganic salt medium containing 5g L ^-1 glucose as the only carbon source; collect 2 mL of the culture, centrifuge it at 12,000 rpm and 4°C for 10 min, and collect the supernatant as bacteria Culture medium sample: Collect 15 mL of culture, centrifuge at 6,000 rpm and 4°C for 10 min to collect the cells, resuspend in 1 mL of ethanol and extract intracellular metabolites at 90°C, dry thoroughly in a vacuum centrifuge and resuspend in an appropriate amount of distilled water. as bacterial lysate sample.

(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

Add gradient concentrations of L-2-HG (1, 3, 5, 10, 30, 50, 70 μM) to the bacterial culture medium and bacterial lysate prepared above, and mix it with the EaLHGFR-2 working solution at a volume ratio of 1:3. Mix in a black 96-well plate, react at 30°C for 60 minutes under dark conditions, and then use a fluorescent microplate reader to measure the fluorescence emission intensity at 590 nm under the excitation wavelength of 544 nm; substitute the obtained fluorescence intensity into the EaLHGFR- described in Example 2 The specific L-2-HG concentration of the above sample was calculated from the dose-response curve of 2 pairs of L-2-HG. Take the same sample as above and use a Thermo Ultimate 3000 rapid separation liquid chromatography system (ThermoFisher Scientific, USA) coupled with a Bruker impact HD ESI-Q-TOF mass spectrometer (BrukerDaltonics, USA) with a Chirobiotic R column (250×4.6mm) ( Supelco Analytical Company of the United States) to determine the actual concentration of L-2-HG in the above samples. The comparison of the detection results of bacterial intracellular and extracellular L-2-HG by EaLHGFR-2 and LC-MS/MS is shown in Figure 8. The measurement results of EaLHGFR-2 are compared with the current standard L-2-HG detection method LC- MS/MS is highly consistent, indicating its accuracy in quantitative detection of bacterial intracellular and extracellular L-2-HG.

(3) Application of EaLHGFR-2 in quantitative detection of bacterial intracellular and extracellular L-2-HG

P.putida KT2440 and P.putida KT2440 (ΔlhgO) were cultured to the mid-log phase in an inorganic salt medium containing 5g L ^-1 glucose or 4g L ^-1 glucose + 1g L ^-1 glutaric acid as the carbon source, using Prepare bacterial lysate and bacterial culture medium samples in the same manner as in (1), and use EaLHGFR-2 to determine the specific concentrations of L-2-HG in different samples according to the method in (2). The results are shown in Figure 9. When the lhgO knockout bacteria were cultured with glucose + glutaric acid as the carbon source, the intracellular and extracellular L-2-HG levels were significantly increased, proving that EaLHGFR-2 is suitable for bacterial intracellular, Quantitative detection of extracellular L-2-HG.

Among them, the formula of the carbon source-free inorganic salt culture medium (1L) described in the above steps (1) and (3) is: 1g NH ₄ Cl, 2.26g KH ₂ PO ₄ , 4.1g K ₂ HPO ₄ , 2.24g NaH ₂ PO ₄ ·H ₂ O, 3.34g Na ₂ HPO ₄ , 10 mL metal ion mixed solution, adjust pH to 7.0 with NaOH, and sterilize at 121°C for 20 minutes. Metal ion mixture (1L): 14.8g MgSO ₄ ·7H ₂ O, 550 mg FeSO ₄ ·7H ₂ O, 45 mg MnSO ₄ ·4H ₂ O, 200 μL H ₂ SO ₄ .

Example 4: Application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG

(1) Preparation of bacterial culture medium and bacterial lysate

Human embryonic kidney cells (HEK293FT) were cultured statically in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin for 24 hours at 37°C and 5% _CO2 ; 2 mL of culture was collected The material was mixed with methanol at a volume ratio of 1:1 and centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was collected as a cell culture medium sample; adherent cultured cells were collected and an appropriate amount of extraction solution (methanol: acetonitrile: distilled water) was added. =5:3:1; add 500 μL of extraction solution per 1×10 ⁶ cells), break the cells by repeated freezing and thawing, extract intracellular metabolites, dry thoroughly in a vacuum centrifuge and resuspend in an appropriate amount of distilled water to serve as cells Lysate sample.

(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

Add gradient concentrations of L-2-HG (1, 3, 5, 10, 30, 50, 70 μM) to the cell culture medium and cell lysate prepared above, and mix it with the EaLHGFR-2 working solution at a volume ratio of 1:3. Mix in a black 96-well plate, react at 30°C for 60 minutes under dark conditions, and then use a fluorescent microplate reader to measure the fluorescence emission intensity at 590 nm under the excitation wavelength of 544 nm; substitute the obtained fluorescence intensity into the EaLHGFR- described in Example 2 The specific L-2-HG concentration of the above sample was calculated from the dose-response curve of 2 pairs of L-2-HG. Take another same sample as above and use LC-MS/MS analysis as described in Example 3 to determine the actual concentration of L-2-HG in the above sample. The comparison of intracellular and extracellular L-2-HG detection results between EaLHGFR-2 and LC-MS/MS is shown in Figure 10. The measurement results of EaLHGFR-2 are consistent with the current standard L-2-HG detection method LC- MS/MS is highly consistent, indicating its accuracy in quantitative detection of intracellular and extracellular L-2-HG.

(3) Application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG

HEK293FT cells were cultured statically under 21% _O2 conditions for 24h, then transferred to the ProOx C21 precision oxygen control system, and continued to be cultured under 0.5% _O2 conditions for 24h. Use the same method as in (1) to prepare HEK293FT cells in normal conditions. For cell lysate and cell culture medium samples cultured under oxygen and anoxic conditions, use EaLHGFR-2 to determine the specific concentrations of L-2-HG in different samples according to the method in (2). The results are shown in Figure 11. Hypoxia treatment caused the intracellular and extracellular L-2-HG levels of HEK293FT cells to increase to 2.59 times and 1.53 times respectively, proving that EaLHGFR-2 is suitable for intracellular and extracellular L-2-HG. Quantitative detection of HG.

Example 5: Application of EaLHGFR-2 in quantitative detection of L-2-HG in human serum and urine

(1) Preparation of human serum and urine samples

In accordance with ethical standards, blood from healthy adults was collected in procoagulant tubes through venous blood collection. After leaving it at room temperature for 2 hours, it was centrifuged at 4°C and 3,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter to obtain serum, which was stored at -20°C. spare. Urine from healthy adults was collected directly, filtered through a 0.22 μm membrane, and stored at -20°C for later use. The application of EaLHGFR-2 to quantitatively detect the concentration of L-2-HG in human serum and urine requires deproteinization of the serum and urine, that is, using a commercial perchloric acid-based deproteinization kit and following the kit steps for deproteinization. operate.

(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

Add gradient concentrations of L-2-HG (1, 3, 5, 10, 30, 50, 70 μM) to the serum and urine samples obtained by removing the protein above, and mix it with the EaLHGFR-2 working solution in black at a volume ratio of 1:3 Mix in a 96-well plate, react at 30°C for 60 minutes under dark conditions, and then use a fluorescent microplate reader to measure the fluorescence emission intensity at 590nm under the excitation wavelength of 544nm; substitute the obtained fluorescence intensity into EaLHGFR-2 described in Example 2 The specific L-2-HG concentration of the above sample was calculated from the dose-response curve for L-2-HG. Take another same sample as above and use LC-MS/MS analysis as described in Example 3 to determine the actual concentration of L-2-HG in the above sample. The comparison of the detection results of L-2-HG in human serum and urine by EaLHGFR-2 and LC-MS/MS is shown in Figure 12. The measurement results of EaLHGFR-2 are consistent with the current standard L-2-HG detection method LC- The MS/MS is highly consistent, indicating its accuracy in the quantitative detection of L-2-HG in human body fluids.

(3) Application of EaLHGFR-2 in the quantitative detection of L-2-HG in body fluids of healthy people and renal cancer patients

Collect serum and urine samples from 7 healthy adults and serum and urine samples from 4 kidney cancer patients using the method described in (1); use EaLHGFR-2 to measure body fluids from healthy people and kidney cancer patients according to the method described in (2) L-2-HG concentration within. The results are shown in Figure 13. The serum L-2-HG concentration of healthy people and renal cancer patients was 0.44 μM to 0.92 μM, and there was no significant difference in the serum L-2-HG concentration between the two groups of people; in addition, the urine of healthy people The average concentration of L-2-HG in the urine was 13.398 μM, while the average concentration of L-2-HG in the urine of kidney cancer patients was 42.19 μM, proving that the urine L-2-HG concentration can be used as a biomarker for kidney cancer.

Nucleotide/amino acid sequences involved in the present invention

Amino acid sequence of CAL94536.1(AoL2HGDH)

METVDCVVVGAGVVGLACARAIAASGREVLILERERAFGTGISSRNSEVIHAGLYYPPGSLKARLSVAGGRMLYAYCESRGVAHRRCGKLVVAAQREALPALARIRARALANGVEELLWLEPGAVKEIEPALDSCGALFSPATGIVDSGHGLMLALLGDAERHGATLVLDTPVLGGRAEAGGIVLQTGGEAPMTLQARCVVNAAGLDAVRLAGQLPASSRGLPQAHFAR GVYFSYAGRVPFSHLIYPVPEPGGLGIHLTLDMGGQPRFGPDVEWIDTPDYTVDPARAERFAAAIRKWWPGLEPERLQPAYAGVRPKIVGPGEADADFQIDGPAEHRVPGLINLLGIESPGLTAALAIGEEVARRIAAPQG(SEQID NO.1)

Amino acid sequence of SNU86518.1

MDKVDCVVIGAGVVGLAVARTMAMAGREVVVLESERAIGTGTSSRNSEVIHGGIYYPPGSRKATLCVEGKHRLYEFCASHGVEHRRCGKLIVATTDHQVAELEAIAANARASGVDDLQWLSAAAEVAQREPALHTFGALLSPSTGIVDSGHLMLALQGDAENAGAMLAFEARVTGARVGRAEGIELDVETEGVTSTLLANTVVNSAGLHAVDIARRF DGLASEHIPQRYYAKGSYFTCAQRAPFTHLIYPVPEPGLGVHLTLDLGGQARFGPNVQWVDEIDYTVNPSDGDGFYAAVRRYWPTLADGALQPGYAGIRPKISGPGEPAADFRIDGPAVHGVAGLVNLFGIESPGLTASLAIAEAVRAALS(SEQID NO.2)

S2DJ52.1 amino acid sequence

MDFQVIIIGGGIVGLATGLKIKQRNPNIKVALLEKEEEVAKHQTGNNSGVIHSGLYYKPGSLKAKNCIEGYHELVRFCEEENIPFELTGKVVVATRKEQVPLLNSLLERGLQNGLKGTRSITLDELKHFEPYCAGVAAIHVPQTGIVDYKLVAEKYAEKFQILGGQVFLGHKVIKVETQNTASIIHTSKGSSFSTNLLINCAGLYSDKVA QMNQKESLDVKIIPFRGEYYKIKKEREYLVKNLIYPVPDPNFPFLGVHFTRMMKGGVEAGPNAVLAFKREGYKKSQVNFSELAETLSWPGFQKVASKYWKTGMGELFRSFSKKAFTDALKELIPDIQESDLIEGGAGVRAQACDRTGGLLDDFCIREDQNAIHVLNAPSPAATSSLSIGGTVCEWALKRF(SEQ ID NO.3)

Nucleotide sequence of CAL94536.1(AoL2HGDH) (after codon optimization)

ATGGAAACCGTTGATTGTGTTGTGGTTGGCGCCGGCGTGGTGGGTCTGGCTTGTGCACGCGCCATTGCCGCAAGTGGTCGCGAAGTGCTGATTCTGGAACGCGAACGCGCATTTGGTACCGGTATTAGTAGCCGCAATAGCGAAGTGATTCATGCAGGTCTGTATTATCCGCCGGGTAGTCTGAAAGCCCGTCTGAGCGTGGCCGGTGGCCGCATGCTGTATGCCTATTGCGAAAGCCGCGGCGTTGCCCATCG TCGCTGCGGTAAACTGGTGGTTGCAGCCCAGCGTGAAGCACTGCCGGCCCTGGCTCGTATTCGTGCCCGTGCACTGGCAAATGGCGTGGAAGAACTGCTGTGGCTGGAACCGGGTGCCGTGAAAGAAATTGAACCGGCACTGGATAGCTGTGGTGCACTGTTTAGCCCGGCAACCGGTATTGTTGATAGCCATGGCCTGATGCTGGCCCTGCTGGGCGATGCAGAACGCCATGGTGCAACCCTGGTTCTGGATACACCTGTTCT GGGTGGCCGTGCCGAAGCAGGTGGCATTGTTCTGCAGACCGGTGGTGAAGCACCTATGACCCTGCAGGCCCGTTGTGTTGTTCTGGATGCCGTGCCTGGCCGGTCAGCTGCCTGCAAGTAGCCGCGGTCTGCCGCAGGCCCATTTTGCACGCGGTGTGTATTTTAGTTATGCAGGCCGCGTTCCGTTTAGCCATCTGATTTATCCGGTGCCGGAACCGGGTGGTCTGGGTTCATCTCT GACCCTGGATATGGGCGGCCAGCCGCGCTTTGGTCCGGATGTGGAATGGATTGATACACCTGATTATACCGTGGACCCTGCCCGTGCCGAACGTTTTGCCGCAGCAATTCGTAAATGGTGGCCGGGCCTGGAACCGGAACGCCTGCAGCCTGCATATGCCGGTGTTCGTCCGAAAATTGTTGGTCCGGGCGAAGCCGATGCAGATTTTCAGATTGATGGCCCGGCCGAACATCGCGTTCCGGGTCTGATTAATCTG CTGGGTATTGAAAGTCCGGGCCTGACCGCCGCCCTGGCTATTGGTGAAGAAGTGGCCCGTCGTATTGCAGCACCTCAGGGCTAA(SEQ ID NO.4)

SNU86518.1 nucleotide sequence (after codon optimization)

ATGGATAAGGTGGATTGCGTTGTTATTGGCGCAGGTGTTGTTGGCCTGGCAGTGGCACGTACCATGGCAATGGCCGGTCGCGAAGTTGTGGTGCTGGAAAGCGAACGTGCCATTGGTACCGGTACCAGCAGTCGTAATAGTGAAGTGATTCATGGTGGCATTTATTATCCGCCGGGCAGTCGTAAAGCCACCCTGTGCGTTGAAGGTAAACATCGTCTGTATGAATTTTGCGCAAGTCATGGTGTTGAACATCGTCGCT GCGGTAAACTGATTGTTGCCACCACCGATCATCAGGTTGCAGAACTGGAAGCAATTGCAGCCAATGCACGTGCCAGCGGTGTTGATGATCTGCAGTGGCTGAGCGCAGCAGAAGTTGCCCAGCGTGAACCGGCACTGCATACCTTTGGCGCACTGCTGAGCCCGAGCACCGGCATTGTGGATAGCCATGGTCTGATGCTGGCCCTGCAGGGCGATGCCGAAAATGCCGGCGCAATGCTGGCCTTTTGAAGCCCGCG TGACCGGTGCACGTGTTGGTCGCGCAGAAGGTATTGAACTGGATGTTGAAACCGAAGGCGTGACCAGCACCCTGCTGGCCAATACCGTTGTGAATAGCGCCGGTCTGCATGCAGTGGATATTGCACGCCGCTTTGATGGCCTGGCCAGTGAACATATTCCGCAGCGCTATTATGCCAAAGGTAGTTATTTTACCTGTGCCCAGCGCGCACCTTTTACCCATCTGATTTATCCGGTTCCGGAACCGGGTGGCCTGGGCGTT CATCTGACCCTGGATCTGGGTGGTCAGGCACGTTTTGGTCCGAATGTTCAGTGGGTGGATGAAATTGATTATACCGTTAATCCGAGTGATGGTGATGGTTTTTATGCCGCCGTTCGCCGTTATTGGCCGACCCTGGCCGATGGCGCACTGCAGCCTGGTTATGCAGGCATTCGTCCGAAAATTAGTGGTCCGGGCGAACCGGCCGCCGATTTTCGTATTGATGGCCCGGCCGTGCATGGTGTTGCAGGCCTGGTG AATCTGTTTGGTATTGAAAGTCCGGGTCTGACCGCCAGCCTGGCAATTGCAGAAGCAGTGCGCGCCGCCCTGAGCTAA(SEQ ID NO.5)

S2DJ52.1 nucleotide sequence (after codon optimization)

ATGGATTTTCAGGTTATCATCATCGGTGGTGGTATTGTTGGTCTGGCCACCGGCCTGAAAATTAAACAGCGTAATCCGAACATTAAAGTGGCGCTGCTGGAAAAAGAAGAAGAAGTTGCAAAACATCAGACCGGTAACAATAGTGGTGTTATTCATAGTGGTCTGTATTATAAACCGGGTAGCCTGAAAGCAAAAAATTGTATTGAAGGTTATCATGAACTGGTTCGTTTTTGTGAAGAAGAAAATTCCGTTTGAACTGACCGGTAAA GTTGTTGTTGCAACCCGTAAAGAACAGGTTCCGCTGCTGAATAGCCTGCTGGAACGTGGTCTGCAGAATGGTCTGAAAGGTACCCGTAGCATTACCCTGGATGAACTGAAACATTTTGAACCGTATTGTGCAGGTGTTGCAGCAATTCATGTTCCGCAGACCGGTATTGTTGATTATAAACTGGTTGCAGAAAAATATGCAGAAAAATTTCAGATTCTGGGTGGTCAGGTTTTTCTGGGTCATAAAGTTATTAAAGTTGAAACCCAGA ATACCGCAAGCATTATTCATACCAGCAAAGGTAGCTTTAGCACCAATCCTGCTGATTAATTGTGCAGGTCTGTATAGCGATAAAGTTGCACAGATGAATCAGAAAGAAAGCCTGGAATTCAGAAAATTCCGTTTCGTGGTGAATAAAATTAAAAAAGAACGTGAATATCTGGTTAAAAATCTGATTTATCCGGTTCCGGACCCTAATTTTCCGTTTCTGGGTGTTCATTTTACCCGTATGATGAAAGGTGGTGTTGAAGCAG GTCCGAATGCAGTTCTGGCATTTAAACGTGAAGGTTATAAAAAAAGCCAGGTTAATTTTAGCGAACTGGCAGAAACCCTGAGCTGGCCGGGTTTTCAGAAAGTTGCAAGCAAATATTGGAAAACCGGTATGGGTGAACTGTTTCGTAGCTTTAGCAAAAAAGCATTTACCGATGCACTGAAAGAACTGATTCCGGATTTCAGGAAAGCGATCTGATTGAAGGTGGTGCAGGTGTTTCGTGCACAGGCATGTGATCGTACCGGTGGT CTGCTGGATGATTTTTGTATTCGTGAAGATCAGAATGCAATTCATGTTCTGAATGCACCGAGCCCGGCAGCAACCAGCAGCCTGAGCATTGGTGGTACCGTTTGTGAATGGGCACTGAAACGTTTTTAA (SEQ ID NO. 6)

The above descriptions are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of this application shall be included in the protection scope of this application.

Claims (10)

1. A high catalytic activity L-2-HG dehydrogenase, characterized in that the high catalytic activity L-2-HG dehydrogenase is derived from bacteria including but not limited to Azoarcus olearius BH72, Pandora sp. Any one or more of Pandoraea sputorum NCTC13161 and Indibacter alkaliphilus LW1; Specifically, the L-2-HG dehydrogenase is selected from: (a1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1-3; (a2) A protein that has the same or similar functions by substituting, deleting and/or adding one or more amino acid residues to the amino acid sequence shown in (a1); (a3) A protein that has an identity of 50% or more with the amino acid sequence composition shown in (a1) or (a2) and has the same or similar function as the protein shown in (a1) or (a2). 2. A polynucleotide, characterized in that the polynucleotide can encode the high catalytic activity L-2-HG dehydrogenase according to claim 1. 3. The polynucleotide of claim 2, wherein the polynucleotide has any one of the nucleotide sequences (b1)-(b4): (b1) The nucleotide sequence shown in SEQ ID NO.4-6; (b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1); (b3) A nucleic acid molecule that has 50% or more identity with the nucleotide sequence defined in (b1) or (b2) and encodes the fusion protein; (b4) A nucleotide sequence capable of hybridizing to the nucleotide sequence described in any one of (b1) to (b3) under stringent conditions and encoding a highly catalytically active L-2-HG dehydrogenase with the same function; Further, the polynucleotide is DNA, including cDNA, genomic DNA or recombinant DNA. 4. A recombinant expression vector, characterized in that the recombinant expression vector at least contains the polynucleotide of claim 2 or 3; Further, the recombinant expression vector is obtained by effectively connecting the polynucleotide of claim 2 or 3 to an expression vector, and the expression vector includes a viral vector, plasmid, phage, cosmid or artificial chromosome; further , the expression vector is a plasmid, including pACYCDuet-1 plasmid. 5. A host cell, characterized in that the host cell contains the polynucleotide of claim 2 or 3, the recombinant expression vector of claim 4 or is capable of expressing the high catalytic activity L- 2-HG dehydrogenase. 6. The high catalytic activity L-2-HG dehydrogenase of claim 1, the polynucleotide of claim 2 or 3, the recombinant expression vector of claim 4 and/or the host cell of claim 5 Application in the preparation of biosensors for detecting L-2-HG. 7. A biosensor for detecting L-2-HG, characterized in that the biosensor at least contains the highly catalytically active L-2-HG dehydrogenase of claim 1 and a redox reaction indicator; wherein, The redox reaction indicator is an electron acceptor that can accept the electrons generated by the dehydrogenation of L-2-HG catalyzed by the highly catalytically active L-2-HG dehydrogenase; Further, the electron acceptor includes azophloroglucinol, 3'-[1-[(anilino)-carbonyl]-3,4-tetrazole]-bis(4-methoxy-6-nitro base) benzene-sodium sulfonate and dichlorophenol indophenol. 8. The biosensor according to claim 7, wherein the biosensor also includes other reagents, devices and/or equipment for L-2-HG detection; Further, the reagent includes a detection buffer; Further, the biosensor for detecting L-2-HG at least includes: 0.1 to 50 μM resazurin, 0.005 to 1 mg mL ^-1 AoL2HGDH, and buffer; The buffer is selected from Tris-HCl, HEPES, potassium phosphate, sodium phosphate, MOPS and PBS, the concentration of the buffer is 50-100mM, and the pH is 5.8-9.0; Further preferably, the biosensor for detecting L-2-HG includes: 67mM PBS (pH 6.6), 1μM resazurin, 0.05mg mL ^-1 AoL2HGDH. 9. A detection kit, characterized in that the detection kit contains the above-mentioned biosensor for detecting L-2-HG. 10. A method for detecting L-2-HG, characterized in that the method at least includes: incubating the sample to be tested with the biosensor according to claim 7 or 8, analyzing the concentration of L-2-HG or none; Further, the sample to be tested is a sample containing or suspected of containing L-2-HG. The sample is a biological sample or an environmental sample. The biological sample includes bacterial culture medium, bacterial lysate, cell culture medium, and cell lysis. fluid, animal serum, animal urine and animal tissue fluid; Furthermore, the animal is a mammal, and the mammal includes a human.