CN116949001A - L-2-hydroxyglutarate biosensor based on high catalytic activity L-2-hydroxyglutarate dehydrogenase and application thereof - Google Patents
L-2-hydroxyglutarate biosensor based on high catalytic activity L-2-hydroxyglutarate dehydrogenase and application thereof Download PDFInfo
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- CN116949001A CN116949001A CN202310936644.0A CN202310936644A CN116949001A CN 116949001 A CN116949001 A CN 116949001A CN 202310936644 A CN202310936644 A CN 202310936644A CN 116949001 A CN116949001 A CN 116949001A
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Abstract
The application belongs to the technical field of biological detection, and particularly relates to an L-2-hydroxyglutarate biosensor based on high-catalytic-activity L-2-hydroxyglutarate dehydrogenase and application thereof. The application screens out L-2-HG dehydrogenase with high catalytic activity from various strains, and based on the construction of the L-2-HG dehydrogenase with high catalytic activity, the response amplitude reaches 2189.25 +/-26.89%, and the detection lower limit is as low as 0.042 mu M, so that the application can realize quantitative detection of L-2-HG in various bacteria, cell samples and human body fluid, thereby being applicable to various fields such as basic research related to L-2-HG, detection and screening of L-2-HG related diseases (such as renal carcinoma) and L-2-HG related drugs (such as L-2-HG activators or inhibitors), and the like, and has wide application prospect.
Description
Technical Field
The application belongs to the technical field of biological detection, and particularly relates to an L-2-hydroxyglutarate biosensor based on high-catalytic-activity L-2-hydroxyglutarate dehydrogenase and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
L-2-Hydroxyglutarate (L-2-HG) is a structural analogue of 2-ketoglutarate (2-KG) in the TCA cycle. Studies have shown that L-2-HG exerts diverse biological functions in normal physiological metabolism of living bodies, including participation in L-lysine decomposition and carbon starvation response, hypoxia adaptation, early embryo development, immune response, and the like. In mammals and microorganisms, L-2-HG is decomposed by a flavoprotein L-2-HG dehydrogenase (called L2HGDH in mammals and LhgO in microorganisms) having FAD as a cofactor, and is converted into 2-KG. The L2HGDH mutation can cause abnormal accumulation of L-2-HG, which leads to altered methylation of histones and DNA in the genome by inhibiting the activity of various 2-KG-dependent dioxygenases, and prevents cell differentiation, eventually leading to the occurrence of cancer, and accumulation of L-2-HG has been found in various diseases such as L-2-hydroxyglutarate, renal cancer, pancreatic cancer and colorectal cancer.
The L-2-HG is a key signal and immune metabolite in a living body, can be used as a key biomarker for diagnosis and prognosis of L-2-HG related diseases, and has important scientific value and clinical significance in developing an accurate, convenient, high-throughput and low-cost L-2-HG detection method. The detection of the L-2-HG mainly depends on the technical means such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), gas chromatography-tandem mass spectrometry (GC-MS/MS), magnetic Resonance Spectroscopy (MRS) and the like, and the methods are expensive, tedious, time-consuming and difficult to realize the separation of the L-2-HG from the enantiomer D-2-hydroxyglutarate (D-2-HG), require professional experimental operators and are incompatible with high-throughput analysis, thus limiting the development of diagnosis and treatment of L-2-HG related diseases and functional diversity research. In addition, a method based onL-2-HG specific transcription regulatory factors and a fluorescent resonance energy transfer technology. Optimized sensor LHGFR 0N3C And LHGFR 0N7C Maximum fluorescence signal change (. DELTA.R) for L-2-HG max I.e., response amplitude) were 56.13 ±0.29% and 60.37 ±1.30%, respectively. In addition, LHGFR 0N3C And LHGFR 0N7C The lower limit of detection (LOD) for L-2-HG in serum was 5.84. Mu.M and 1.68. Mu.M, respectively, and the lower limit of detection for L-2-HG in urine was 15.74. Mu.M and 0.92. Mu.M, respectively. The lower response amplitude and the higher detection lower limit the LHGFR in the aspect of accurate quantification of low-concentration L-2-HG, such as the inability to detect L-2-HG in body fluid of healthy adults. Therefore, there is a 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 requirements of quantitative detection of L-2-HG.
Disclosure of Invention
In order to overcome the defects in the prior art, the inventor provides an L-2-HG biosensor based on high catalytic activity L-2-HG dehydrogenase and application thereof through long-term technical and practical exploration. The application screens out L-2-HG dehydrogenase with high catalytic activity from various strains, and constructs the L-2-HG biosensor based on the L-2-HG dehydrogenase with high catalytic activity. Experiments prove that the response amplitude of the L-2-HG biosensor reaches 2189.25 +/-26.89%, and the detection lower limit is as low as 0.042 mu M, so that quantitative detection of various bacteria, cell samples and L-2-HG in human body fluid can be realized. Based on the above results, the present application has been completed.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect of the present application, there is provided a high catalytic activity L-2-HG dehydrogenase derived from any one or more of the group consisting of, but not limited to, vibrio azotemlobus (Azoarcus olearius) BH72, pandora (Pandoraea sputorum) NCTC13161, and Indian bacillus (Indibacter alkaliphilus) LW 1.
Specifically, the L-2-HG dehydrogenase is selected from the group consisting of:
(a1) A protein consisting of the amino acid sequence shown in any one of SEQ ID NO. 1-3;
(a2) A protein having the same or similar function by substituting, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in (a 1);
(a3) A protein having an amino acid sequence composition identical to that of (a 1) or (a 2) of 50% or more and having the same or similar function to that of the protein of (a 1) or (a 2).
In a second aspect of the present application, there is provided a polynucleotide capable of encoding the above-described highly catalytically active L-2-HG dehydrogenase.
Specifically, the polynucleotide has the nucleotide sequence of any one of (b 1) to (b 4):
(b1) Nucleotide sequence shown as SEQ ID NO. 4-6;
(b2) A sequence formed by substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence as shown in (b 1);
(b3) A nucleic acid molecule having 50% or more identity to the nucleotide sequence defined in (b 1) or (b 2) and encoding said fusion protein;
(b4) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence according to any one of (b 1) to (b 3) and which encodes the same functional, high catalytic activity L-2-HG dehydrogenase.
In a third aspect of the application, there is provided a recombinant expression vector comprising at least a polynucleotide as described above.
In a fourth aspect of the present application, there is provided a host cell comprising the above polynucleotide, the above recombinant expression vector or capable of expressing the above highly catalytically active L-2-HG dehydrogenase.
In a fifth aspect, the present application provides the use of the above-described highly catalytically active L-2-HG dehydrogenase, polynucleotide, recombinant expression vector and/or cell in the preparation of a biosensor for detecting L-2-HG.
In a sixth aspect of the present application, there is provided a biosensor (which may be named as EaLHGFR) for detecting L-2-HG, which comprises at least the above-mentioned high catalytic activity L-2-HG dehydrogenase, and a redox reaction indicator; wherein the redox indicator may be an electron acceptor that accepts the dehydrogenation of L-2-HG to produce electrons by the L-2-HG dehydrogenase.
The biosensor can exist in the form of a detection kit in the practical application process.
In a seventh aspect of the present application, there is provided a method of detecting L-2-HG, the method comprising at least: and incubating a sample to be tested with the biosensor, and analyzing the concentration or presence of the L-2-HG.
Compared with the prior art, the one or more technical schemes have the following beneficial technical effects:
(1) The technical scheme is based on sequence comparison analysis, and a plurality of L-2-HG dehydrogenases with high catalytic activity are screened from different strains; wherein, L-2-HG dehydrogenase AoL HGDH with high substrate specificity and high catalytic activity is identified in A.olearius BH 72; aoL2HGDH takes FAD as a cofactor, can directly transfer electrons to the redox reaction indicator while catalyzing the conversion of L-2-HG to 2-KG, and does not need an additional electron transfer mediator;
(2) The L-2-HG biosensor EaLHGFR based on the high catalytic activity L-2-HG dehydrogenase, which is developed by the technical scheme, has the characteristics of high response amplitude and low detection lower limit, and the detection system only comprises AoL HGDH and a redox reaction indicator, so that the preparation is simple, the components are simple, the cost is low, the operation is easy, and the high-flux detection can be realized;
(3) The L-2-HG biosensor EaLHGFR based on the L-2-HG dehydrogenase with high catalytic activity, which is developed by the technical scheme, is suitable for quantitative detection of L-2-HG in biological samples such as different bacteria, cells, human serum, urine and the like, and the detection result is highly consistent with the LC-MS/MS of the current L-2-HG standard detection method, thus having wide application prospect in the detection of L-2-HG in various biological samples.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application.
FIG. 1 is a comparison of enzyme activities of L-2-HG dehydrogenases of different origins in example 1 of the present application.
FIG. 2 shows SDS-PAGE verification of AoL HGDH expression purification in example 1 of the present application.
FIG. 3 is a kinetic parameter determination and substrate specificity analysis of AoL HGDH to L-2-HG in example 1 of the present application; wherein A is the kinetic parameter measurement of AoL2HGDH to L-2-HG, and B is the substrate specificity analysis of AoL HGDH.
FIG. 4 is a schematic diagram showing the detection principle of an L-2-HG biosensor based on AoL HGDH in example 2 of the present application.
FIG. 5 is a graph showing the dose-response of the initial L-2-HG biosensor EaLHGFR-1 to L-2-HG in example 2 of the present application.
FIG. 6 is a system optimization of the L-2-HG biosensor in example 2 of the present application.
FIG. 7 is a graph showing the dose-response of the optimized L-2-HG biosensor EaLHGFR-2 to L-2-HG in example 2 of the present application.
FIG. 8 is a consistency analysis of EaLHGFR-2 with LC-MS/MS for detection of bacterial samples L-2-HG in example 3 of the present application; wherein A is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detection of L-2-HG in bacterial lysates, and B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detection of L-2-HG in bacterial culture media.
FIG. 9 shows the quantitative detection of intracellular and extracellular L-2-HG of bacteria by using EaLHGFR-2 in example 3 of the present application; wherein A is the result of quantitative detection of L-2-HG in bacterial cells, and B is the result of quantitative detection of L-2-HG out of bacterial cells.
FIG. 10 is a consistency analysis of EaLHGFR-2 with LC-MS/MS for detection of cell samples L-2-HG in example 4 of the present application; wherein A is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detection of L-2-HG in cell lysates, and B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detection of L-2-HG in cell culture media.
FIG. 11 shows the quantitative detection of intracellular and extracellular L-2-HG by using EaLHGFR-2 in example 4 of the present application; wherein A is the result of quantitative detection of intracellular L-2-HG, and B is the result of quantitative detection of extracellular L-2-HG.
FIG. 12 is a consistency analysis of EaLHGFR-2 and LC-MS/MS for human body fluid L-2-HG detection in example 5 of the present application; wherein A is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detecting L-2-HG in human serum, and B is the consistency analysis of EaLHGFR-2 and LC-MS/MS for detecting L-2-HG in human urine.
FIG. 13 shows the quantitative detection of L-2-HG in human body fluid by using EaLHGFR-2 in example 5 of the present application; wherein A is the result of quantitative detection of human serum L-2-HG, and B is the result of quantitative detection of human urine L-2-HG.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, 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 belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In one exemplary embodiment of the present application, a high catalytic activity L-2-HG dehydrogenase is provided, which is derived from any one or more of the group consisting of, but not limited to, vibrio azotemmaum (Azoarcus olearius) BH72, pandora (Pandoraea sputorum) NCTC13161, and Indian bacillus (Indibacter alkaliphilus) LW 1; further derived from Vibrio azotemma (Azoarcus olearius) BH72, this highly catalytically active L-2-HG dehydrogenase was designated AoL2HGDH, geneBank accession number CAL94536.1; aoL2HGDH covalently binds FAD as cofactor and catalyzes the production of 2-KG from L-2-HG, and has high substrate specificity and high dehydrogenation activity to L-2-HG.
The L-2-HG dehydrogenase is selected from the group consisting of:
(a1) Proteins consisting of the amino acid sequences shown in SEQ ID NO. 1-3;
(a2) A protein having the same or similar function by substituting, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in (a 1);
(a3) A protein having an amino acid sequence composition identical to that of (a 1) or (a 2) of 50% or more and having the same or similar function to that of the protein of (a 1) or (a 2).
Wherein in said (a 2), the substitution, deletion and/or addition of said one or more amino acid residues is typically a substitution and/or deletion and/or addition of not more than 15 amino acid residues.
The proteins shown in the above (a 1) to (a 3) may be synthesized artificially or may be obtained by synthesizing the genes encoding them and then biologically expressing them.
In one or more embodiments of the present application, a polynucleotide is provided that encodes the high catalytic activity L-2-HG dehydrogenase described above.
Specifically, the polynucleotide has the nucleotide sequence of any one of (b 1) to (b 4):
(b1) Nucleotide sequence shown as SEQ ID NO. 4-6;
(b2) A sequence formed by substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence as shown in (b 1);
(b3) A nucleic acid molecule having 50% or more identity to the nucleotide sequence defined in (b 1) or (b 2) and encoding said fusion protein;
(b4) A nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence according to any one of (b 1) to (b 3) and which encodes the same functional, high catalytic activity L-2-HG dehydrogenase.
It is noted that the term "identity" refers to sequence similarity to amino acid/nucleotide sequences. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The above 50% identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% identity.
The polynucleotide may be DNA such as cDNA, genomic DNA, recombinant DNA, or the like, and is not particularly limited herein.
In one or more embodiments of the present application, there is provided a recombinant expression vector comprising at least the above polynucleotide.
The recombinant expression vector is obtained by operably linking the above polynucleotide to an expression vector, including a viral vector (including an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector), a plasmid, a phage, a cosmid, or an artificial chromosome; further, the expression vector may be a plasmid, such as pACYCDuet-1 plasmid.
In one or more embodiments of the present application, a host cell is provided, which comprises the above polynucleotide, the above recombinant expression vector, or is capable of expressing the above highly catalytically active L-2-HG dehydrogenase.
The host cell includes a bacterial cell or a fungal cell;
the bacteria may be any one or more of the genera escherichia, agrobacterium, bacillus, streptomyces, pseudomonas or staphylococcus.
In one or more embodiments of the application, the bacterium is E.coli (e.g., BL21 (DE 3)), agrobacterium tumefaciens (e.g., GV 3101), agrobacterium rhizogenes, lactococcus lactis, bacillus subtilis, bacillus cereus, or Pseudomonas fluorescens.
In one or more embodiments of the present application, there is provided the use of the above-described highly catalytically active L-2-HG dehydrogenase, polynucleotide, recombinant expression vector and/or cell in the preparation of a biosensor for detecting L-2-HG.
In one or more embodiments of the present application, there is provided a biosensor (which may be named as EaLHGFR) for detecting L-2-HG, which comprises at least the above-mentioned high catalytic activity L-2-HG dehydrogenase, and a redox reaction indicator; wherein the redox indicator may be an electron acceptor that accepts the dehydrogenation of L-2-HG to produce electrons by the L-2-HG dehydrogenase.
The electron acceptors include, but are not limited to, azure, sodium 3' - [1- [ (anilino) -carbonyl ] -3, 4-tetrazole ] -bis (4-methoxy-6-nitro) benzene-sulfonate, and dichlorophenol indophenol.
The biosensor may also include other reagents, devices and/or apparatus for L-2-HG detection;
the reagent includes a detection buffer.
In still another embodiment of the present application, the biosensor for detecting L-2-HG includes: 0.1-50 mu M of resazurin and 0.005-1 mg mL -1 AoL2HGDH and buffers; the buffer solution is selected from Tris-HCl, HEPES, potassium phosphate, sodium phosphate, MOPS and PBS, the concentration of the buffer solution is 50-100 mM, and the pH value is 5.8-9.0;
further preferably, the biosensor for detecting L-2-HG includes: 67mM PBS (pH 6.6), 1. Mu.M resazurin, 0.05mg mL -1 AoL2HGDH. Under such conditions, when L-2-HG is present in the reaction system, aoL HGDH catalyzes the dehydrogenation of L-2-HG, accompanied by the conversion of covalently bound FAD into FADH 2 ，FADH 2 The electrons are further transferred to the resazurin to generate the high red-shift fluorescent molecule resorufin (emission peak is at 587 nm), and at the moment, the high red-shift fluorescent molecule resorufin has the highest response amplitude to 0.5 mu M and 1 mu M L-2-HG, and the response amplitude is 2189.25 +/-26.89% and the detection lower limit is 0.042 mu M through test verification, so that the high-sensitivity detection of the L-2-HG is realized.
The biosensor can exist in the form of a detection kit in the practical application process.
The kit may comprise materials or reagents (including highly catalytically active L-2-HG dehydrogenase, redox indicators, etc.) used in carrying out the methods of the present application. The kit may include storage reagents (e.g., L-2-HG dehydrogenase, redox indicators, etc. in a suitable container) and/or support materials (e.g., buffers, instructions for performing the assay, etc.). For example, the kit may comprise one or more containers (e.g., cassettes) containing the respective 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, a second container contains the redox reaction indicator, and a third container contains the buffer. The kit may also contain a compartment suitable for holding the reagents or containers. The present application is not particularly limited herein.
In one or more embodiments of the present application, there is provided a method of detecting L-2-HG, the method including at least: and incubating a sample to be tested with the biosensor, and analyzing the concentration or presence of the L-2-HG.
Wherein the sample to be tested is a sample containing or suspected of containing L-2-HG, which may be a biological sample or an environmental sample, including but not limited to bacterial culture medium, bacterial lysate, cell culture medium, cell lysate, animal serum, animal urine, and animal tissue fluid;
wherein the animal may be a mammal, and wherein a human is preferred.
The qualitative or quantitative detection of the L-2-HG can be applied to various fields such as L-2-HG related basic research, detection and screening of L-2-HG related diseases (such as renal carcinoma) and L-2-HG related medicaments (such as L-2-HG activators or inhibitors) and the like, and therefore, the method has wide application prospect.
The application is further illustrated by the following examples, which are given for the purpose of illustration only and are not intended to be limiting. The experimental methods used, not specifically described, are all conventional methods. The strains, cells, materials, reagents and the like used, unless otherwise specified, are all commercially available.
Example 1: identification and characterization of high catalytic activity L-2-HG dehydrogenase AoL HGDH protein
(1) Exogenous expression and enzyme activity comparison of different L-2-HG dehydrogenase homologous proteins
Based on sequence comparison analysis, obtaining the nucleotide sequences of the L-2-HG dehydrogenase homologous proteins of ten bacteria, fungi and plant sources, and entrusting the synthesis of general biological systems (Anhui) limited companyThe strain is inoculated into pACYCDuet-1 plasmid and then preserved in E.coli BL21 (DE 3); the expression strains were inoculated with chloramphenicol (40. Mu.g mL) -1 ) Is cultured to OD at 37 ℃ and 180rpm 600nm About 0.6, 1mM IPTG was added and protein expression was induced overnight at 23℃and 160 rpm; centrifugally collecting thalli, and crushing under high pressure to obtain crude enzyme liquid of each L-2-HG dehydrogenase; the activity of each L-2-HG dehydrogenase on L-2-HG was measured in 800. Mu.L of a reaction system containing 50mM Tris-HCl (pH 7.4), 100. Mu.M DCPIP, 100. Mu.M L-2-HG and 40. Mu.L of crude enzyme solution, and the catalytic activity of each L-2-HG dehydrogenase on L-2-HG was determined by detecting the change in absorbance at 600nm at 30 ℃. The results are shown in FIG. 1, wherein the L-2-HG dehydrogenase derived from A.olearius BH72 (GeneBank: CAL 94536.1) has the highest catalytic activity on L-2-HG, and is named AoL HGDH, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO. 4.
(2) Expression purification of AoL2HGDH
E.coli BL21 (DE 3) strain carrying AoL HGDH expression vector was activated in LB liquid medium for two generations and inoculated into a medium containing chloramphenicol (40. Mu.g mL) -1 ) In 500mL LB liquid medium, at 37℃and 180rpm to OD 600nm About 0.6, 1mM IPTG was added and AoL HGDH expression was induced overnight at 23℃and 160 rpm; the cells were collected by centrifugation, washed twice with binding buffer and resuspended to OD 600nm 20, adding 1mM PMSF and 10% glycerol, crushing the thalli under high pressure, centrifuging at 12,000rpm and 4 ℃ for 50min to remove cell debris, and obtaining a crude enzyme solution of AoL HGDH; filtering the obtained crude enzyme solution by a filter head with the thickness of 0.22 mu m, separating and purifying by using a 5mL nickel column, and eluting by using elution buffers with different concentrations to obtain purified AoL HGDH; the purity of AoL2HGDH was checked by SDS-PAGE, and the results are shown in FIG. 2.
(3) Kinetic parameter determination and substrate specificity analysis of AoL2HGDH
The kinetic parameters of AoL HGDH to L-2-HG were determined in 800. Mu.L reaction system containing 50mM Tris-HCl (pH 7.4), 100. Mu.M DCPIP, gradient concentration L-2-HG and appropriate amount of purified AoL HGDH protein, by detecting the change in absorbance at 600nm at 30 ℃AoL2 catalytic activity of HGDH to gradient concentration L-2-HG, fitting calculation of kinetic parameters of AoL HGDH to L-2-HG; the results are shown in FIG. 3A, aoL2HGDH vs. L-2-HG max The value is 50.85 +/-1.66U mg -1 、K m The value was 29.12.+ -. 2.52. Mu.M. The substrate specificity analysis reaction system of AoL HGDH is the same as that of the reaction system, namely, only L-2-HG is replaced by 10 mu M of L-2-HG, L-tartaric acid, D-tartaric acid, L-glyceric acid, D-glyceric acid, L-lactic acid, D-lactic acid and other L-2-HG structural analogues, and the substrate specificity of the reaction system is determined by measuring the activity of AoL HGDH on the different structural analogues; as a result, as shown in FIG. 3B, aoL2HGDH had high specificity for L-2-HG.
Wherein, the formula of the LB medium in the steps (1) - (2) is as follows: yeast powder 5g L -1 The method comprises the steps of carrying out a first treatment on the surface of the Peptone 10g L -1 ；NaCl 10g L -1 pH 7.0; sterilizing at 121deg.C for 20min.
The formula of the binding buffer solution in the step (2) is as follows: 20mM Na 2 HPO 4 20mM imidazole, 500mM NaCl, pH 7.4; the elution buffer formulation was: 20mM Na 2 HPO 4 The pH was adjusted to 7.4 with 500mM imidazole, 500mM NaCl.
Example 2: construction and optimization of L-2-HG biosensor based on high catalytic activity L-2-HG dehydrogenase AoL2HGDH
(1) Construction principle of L-2-HG biosensor based on high catalytic activity L-2-HG dehydrogenase AoL2HGDH
AoL2HGDH has high catalytic activity and high substrate specificity, can be used as an ideal catalytic element to be combined with a resazurin-mediated redox report system, and develops an L-2-HG biosensor (EaLHGFR) based on an enzymatic method. EaLHGFR assay is shown in FIG. 4, in which AoL HGDH catalyzes the dehydrogenation of L-2-HG to 2-KG, while reducing covalently bound FAD to FADH 2 The method comprises the steps of carrying out a first treatment on the surface of the FADH produced 2 The resazurin can be further reduced to generate the high red-shift fluorescent molecule resorufin.
(2) Construction and characterization of the initial L-2-HG biosensor EaLHGFR-1
Preparation of 50mM Tris-HCl (pH 7.4), 15. Mu.M resazurin, 0.1mg mL -1 AoL2 HGDH-constructed L-2-HG biosensor working solution (initial)The L-2-HG biosensor was designated as EaLHGFR-1), a gradient concentration L-2-HG solution (0 mM to 20 mM) was prepared, 75. Mu.L of LHGFR-1 working solution was mixed with 25. Mu.L of L-2-HG solution in a black 96-well plate, reacted at 30℃for 60 minutes under a dark condition, and the fluorescence emission intensity at 590nm was measured at an excitation wavelength of 544nm using a fluorescence microplate reader. The dose-response curve of EaLHGFR-1 versus L-2-HG is plotted with the concentration of L-2-HG on the abscissa and the fluorescence intensity of EaLHGFR-1 on the ordinate. As shown in FIG. 5, eaLHGFR-1 responds in a dose-dependent manner to the addition of L-2-HG with a response amplitude (i.e., maximum fluorescence intensity variation, ΔF max ) 1933.73.+ -. 38.99% and the lower limit of detection is 0.976. Mu.M.
(3) System optimization of L-2-HG biosensor
To increase the detection sensitivity of the L-2-HG biosensor, a single factor screening strategy was used for the detection buffer type (50 mM 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]67mM PBS [ pH 7.4 ]]) The pH of the assay buffer (5.8, 6.2, 6.6, 7.0, 7.4, 7.8, 8.2, 8.6, 9.0), the concentration of Resazurin (0.1, 0.5, 1, 5, 10, 15, 50. Mu.M) and the concentration of AoL2HGDH (0.005, 0.01, 0.05, 0.07, 0.1, 0.5, 1mg mL) -1 ) The detection sensitivity of the biosensor was determined by the response amplitude of the biosensor to low concentrations of L-2-HG (0.5. Mu.M and 1. Mu.M) by performing system optimization. As a result, as shown in FIG. 6, the sample was prepared from 67mM PBS (pH 6.6), 1. Mu.M resazurin, and 0.05mg mL -1 AoL2HGDH constructed biosensor variant with highest response amplitude to 0.5. Mu.M and 1. Mu.M L-2-HG was increased from 29% and 66% of EaLHGFR-1 to 488% and 885%, respectively, and the sensor variant was named EaLHGFR-2.
(4) Characterization of the properties of the optimized L-2-HG biosensor EaLHGFR-2
The dose-response curve of EaLHGFR-2 versus L-2-HG was determined as described in (2). As a result, as shown in FIG. 7, eaLHGFR-2 responds to the addition of L-2-HG in a dose-dependent manner with a response amplitude of 2189.25.+ -. 26.89% and a detection limit as low as 0.042. Mu.M.
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
The P.putida KT2440 strain was isolated in the presence of 5. 5g L -1 Culturing in an inorganic salt culture medium with glucose as the sole carbon source to mid-log phase; 2mL of the culture was collected, centrifuged at 12,000rpm at 4℃for 10min, and the supernatant was collected as a bacterial culture medium sample; 15mL of the culture was collected, centrifuged at 6,000rpm at 4℃for 10min to collect the cells, resuspended in 1mL of ethanol, and the intracellular metabolites were extracted at 90℃and thoroughly dried in a vacuum centrifuge and resuspended in an appropriate amount of distilled water as a bacterial lysate sample.
(2) Detection consistency analysis of EaLHGFR-2 and LC-MS/MS
Adding gradient concentration L-2-HG (1, 3, 5, 10, 30, 50, 70 mu M) into the prepared bacterial culture medium and bacterial lysate, mixing with EaLHGFR-2 working solution in a black 96-well plate according to a volume ratio of 1:3, reacting for 60min at 30 ℃ under a dark condition, and measuring fluorescence emission intensity at 590nm under an excitation wavelength of 544nm by using a fluorescence microplate reader; the specific L-2-HG concentration of the above sample was calculated by substituting the obtained fluorescence intensity into the dose-response curve of EaLHGFR-2 versus L-2-HG described in example 2. The actual concentration of L-2-HG in the above samples was determined using a Thermo Ultimate 3000 fast separation liquid chromatography system (America ThermoFisher Scientific) in series with a Bruker impact HD ESI-Q-TOF mass spectrometer (Bruker Daltonics, america) along with a Chirobiotic R column (250X 4.6 mm) (America Supelco Analytical). Comparison of EaLHGFR-2 and LC-MS/MS on detection results of bacterial intracellular and extracellular L-2-HG is shown in figure 8, and the detection result of EaLHGFR-2 is highly consistent with the current standard L-2-HG detection method LC-MS/MS, so that the accuracy of the detection method applied to quantitative detection of bacterial intracellular and extracellular L-2-HG is shown.
(3) Application of EaLHGFR-2 in quantitative detection of bacterial intracellular and extracellular L-2-HG
P.putida KT2440 and P.putida KT2440 (. DELTA.lhgO) were mixed at a concentration of 5g L -1 Glucose or 4g L -1 Glucose +1g L -1 Culturing in inorganic salt culture medium with glutaric acid as carbon source to mid-log phase, and preparing bacteria by the same method as in (1)Lysates and bacterial culture samples specific concentrations of L-2-HG in the different samples were determined according to method (2) using EaLHGFR-2. As shown in FIG. 9, the levels of intracellular and extracellular L-2-HG are obviously increased when the lhgO knockout bacterium is cultured by taking glucose and glutaric acid as carbon sources, and the EaLHGFR-2 is proved to be suitable for quantitative detection of intracellular and extracellular L-2-HG of bacteria.
Wherein, the formula of the inorganic salt culture medium (1L) without carbon source in the steps (1) and (3) is as follows: 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 10mL of the metal ion mixed solution, pH was adjusted to 7.0 with NaOH, and sterilization was performed at 121℃for 20 minutes. Metal ion mixed solution (1L): 14.8g MgSO 4 ·7H 2 O，550mg FeSO 4 ·7H 2 O，45mg MnSO 4 ·4H 2 O，200μL H 2 SO 4 。
Example 4: application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG of cell
(1) Preparation of bacterial culture Medium and bacterial lysate
Human embryonic kidney cells (HEK 293 FT) were cultured in high-sugar DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37deg.C and 5% CO 2 Standing and culturing for 24 hours under the condition of (2); 2mL of culture was collected, mixed with methanol at a volume ratio of 1:1, and centrifuged at 12,000rpm at 4℃for 10min, and the supernatant was collected as a cell culture medium sample; the cells cultured by adherence were collected, and an appropriate amount of the extract (methanol: acetonitrile: distilled water=5:3:1; every 1×10) was added 6 Adding 500 mu L of extract into each cell) to break up the cells by repeated freeze thawing, extracting intracellular metabolites, and re-suspending the cells by using a proper amount of distilled water after thoroughly drying the cells by a vacuum centrifuge to obtain a cell lysate sample.
(2) Detection consistency analysis of EaLHGFR-2 and LC-MS/MS
Adding gradient concentration L-2-HG (1, 3, 5, 10, 30, 50, 70 mu M) into the prepared cell culture medium and cell lysate, mixing with EaLHGFR-2 working solution in a black 96-well plate according to a volume ratio of 1:3, reacting for 60min at 30 ℃ under a dark condition, and measuring fluorescence emission intensity at 590nm under an excitation wavelength of 544nm by using a fluorescence microplate reader; the specific L-2-HG concentration of the above sample was calculated by substituting the obtained fluorescence intensity into the dose-response curve of EaLHGFR-2 versus L-2-HG described in example 2. The same samples were taken and the actual concentration of L-2-HG in the samples was determined using LC-MS/MS analysis in the manner described in example 3. Comparison of EaLHGFR-2 and LC-MS/MS on intracellular and extracellular L-2-HG detection results is shown in figure 10, and the EaLHGFR-2 detection results are highly consistent with the current standard L-2-HG detection method LC-MS/MS, so that the accuracy of the method applied to quantitative detection of intracellular and extracellular L-2-HG of cells is shown.
(3) Application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG of cell
HEK293FT cells were grown at 21% O 2 Stationary culturing under the condition for 24h, transferring into ProOx C21 precise oxygen control system, and adding 0.5% O 2 The culture was continued for 24 hours using the same method as in (1) to prepare a cell lysate and a cell culture medium sample of HEK293FT under normoxic and anoxic conditions, respectively, and specific concentrations of L-2-HG in the different samples were determined using EaLHGFR-2 according to the method in (2). As shown in FIG. 11, the hypoxia treatment resulted in an increase in intracellular and extracellular L-2-HG levels of HEK293FT cells by a factor of 2.59 and 1.53, respectively, demonstrating that EaLHGFR-2 is suitable for quantitative detection of intracellular and extracellular L-2-HG.
Example 5: application of EaLHGFR-2 in quantitative detection of human serum and urine L-2-HG
(1) Human serum and urine sample preparation
According to ethical specifications, blood of a healthy adult is collected by a venous blood collection method in a coagulation promoting tube, placed at room temperature for 2 hours, centrifuged at 3,000rpm for 10 minutes at 4 ℃, and the supernatant is filtered through a 0.22 μm filter membrane to obtain serum, which is stored at-20 ℃ for later use. Directly collecting urine of healthy adult, filtering with 0.22 μm filter membrane, and storing at-20deg.C. The concentration of L-2-HG in human serum and urine is quantitatively detected by using EaLHGFR-2, and the serum and urine are subjected to deproteinization treatment, namely, a commercial deproteinization kit based on perchloric acid is adopted, and deproteinization operation is carried out according to the steps of the kit.
(2) Detection consistency analysis of EaLHGFR-2 and LC-MS/MS
Adding gradient concentration L-2-HG (1, 3, 5, 10, 30, 50, 70 mu M) into serum and urine samples obtained by removing protein, mixing with EaLHGFR-2 working solution in a black 96-well plate according to a volume ratio of 1:3, reacting for 60min at 30 ℃ under a light-shielding condition, and measuring fluorescence emission intensity at 590nm under an excitation wavelength of 544nm by using a fluorescence microplate reader; the specific L-2-HG concentration of the above sample was calculated by substituting the obtained fluorescence intensity into the dose-response curve of EaLHGFR-2 versus L-2-HG described in example 2. The same samples were taken and the actual concentration of L-2-HG in the samples was determined using LC-MS/MS analysis in the manner described in example 3. The comparison of EaLHGFR-2 and LC-MS/MS on the detection results of L-2-HG in human serum and urine is shown in figure 12, and the detection result of EaLHGFR-2 is highly consistent with the current standard L-2-HG detection method LC-MS/MS, which shows the accuracy of the quantitative detection of the L-2-HG applied to human body fluid.
(3) Application of EaLHGFR-2 in quantitative detection of body fluid L-2-HG of healthy people and kidney cancer patients
Collecting serum and urine samples of 7 healthy adults and serum and urine samples of 4 patients with renal cancer by the method described in (1); the concentration of L-2-HG in the body fluid of healthy persons and kidney cancer patients was measured by the method of (2) using EaLHGFR-2. As shown in FIG. 13, serum L-2-HG of healthy people and kidney cancer patients is 0.44 mu M to 0.92 mu M, and serum L-2-HG concentration of two groups of people is not significantly different; in addition, the average concentration of L-2-HG in urine of healthy people is 13.398 mu M, and the average concentration of L-2-HG in urine of patients with renal cancer is 42.19 mu M, which proves that the concentration of the urine L-2-HG can be used as a biomarker of renal cancer.
Nucleotide/amino acid sequences involved in the present application
Amino acid sequence of CAL94536.1 (AoL HGDH)
METVDCVVVGAGVVGLACARAIAASGREVLILERERAFGTGISSRNSEVIHAGLYYPPGSLKARLSVAGGRMLYAYCESRGVAHRRCGKLVVAAQREALPALARIRARALANGVEELLWLEPGAVKEIEPALDSCGALFSPATGIVDSHGLMLALLGDAERHGATLVLDTPVLGGRAEAGGIVLQTGGEAPMTLQARCVVNAAGLDAVRLAGQLPASSRGLPQAHFARGVYFSYAGRVPFSHLIYPVPEPGGLGIHLTLDMGGQPRFGPDVEWIDTPDYTVDPARAERFAAAIRKWWPGLEPERLQPAYAGVRPKIVGPGEADADFQIDGPAEHRVPGLINLLGIESPGLTAALAIGEEVARRIAAPQG(SEQ ID NO.1)
Amino acid sequence of SNU86518.1
MDKVDCVVIGAGVVGLAVARTMAMAGREVVVLESERAIGTGTSSRNSEVIHGGIYYPPGSRKATLCVEGKHRLYEFCASHGVEHRRCGKLIVATTDHQVAELEAIAANARASGVDDLQWLSAAEVAQREPALHTFGALLSPSTGIVDSHGLMLALQGDAENAGAMLAFEARVTGARVGRAEGIELDVETEGVTSTLLANTVVNSAGLHAVDIARRFDGLASEHIPQRYYAKGSYFTCAQRAPFTHLIYPVPEPGGLGVHLTLDLGGQARFGPNVQWVDEIDYTVNPSDGDGFYAAVRRYWPTLADGALQPGYAGIRPKISGPGEPAADFRIDGPAVHGVAGLVNLFGIESPGLTASLAIAEAVRAALS(SEQ ID NO.2)
S2DJ52.1 amino acid sequence
MDFQVIIIGGGIVGLATGLKIKQRNPNIKVALLEKEEEVAKHQTGNNSGVIHSGLYYKPGSLKAKNCIEGYHELVRFCEEENIPFELTGKVVVATRKEQVPLLNSLLERGLQNGLKGTRSITLDELKHFEPYCAGVAAIHVPQTGIVDYKLVAEKYAEKFQILGGQVFLGHKVIKVETQNTASIIHTSKGSFSTNLLINCAGLYSDKVAQMNQKESLDVKIIPFRGEYYKIKKEREYLVKNLIYPVPDPNFPFLGVHFTRMMKGGVEAGPNAVLAFKREGYKKSQVNFSELAETLSWPGFQKVASKYWKTGMGELFRSFSKKAFTDALKELIPDIQESDLIEGGAGVRAQACDRTGGLLDDFCIREDQNAIHVLNAPSPAATSSLSIGGTVCEWALKRF(SEQ ID NO.3)
Nucleotide sequence (after codon optimization) of CAL94536.1 (AoL 2 HGDH)
ATGGAAACCGTTGATTGTGTTGTGGTTGGCGCCGGCGTGGTGGGTCTGGCTTGTGCACGCGCCATTGCCGCAAGTGGTCGCGAAGTGCTGATTCTGGAACGCGAACGCGCATTTGGTACCGGTATTAGTAGCCGCAATAGCGAAGTGATTCATGCAGGTCTGTATTATCCGCCGGGTAGTCTGAAAGCCCGTCTGAGCGTGGCCGGTGGCCGCATGCTGTATGCCTATTGCGAAAGCCGCGGCGTTGCCCATCGTCGCTGCGGTAAACTGGTGGTTGCAGCCCAGCGTGAAGCACTGCCGGCCCTGGCTCGTATTCGTGCCCGTGCACTGGCAAATGGCGTGGAAGAACTGCTGTGGCTGGAACCGGGTGCCGTGAAAGAAATTGAACCGGCACTGGATAGCTGTGGTGCACTGTTTAGCCCGGCAACCGGTATTGTTGATAGCCATGGCCTGATGCTGGCCCTGCTGGGCGATGCAGAACGCCATGGTGCAACCCTGGTTCTGGATACACCTGTTCTGGGTGGCCGTGCCGAAGCAGGTGGCATTGTTCTGCAGACCGGTGGTGAAGCACCTATGACCCTGCAGGCCCGTTGTGTTGTTAATGCAGCAGGTCTGGATGCCGTGCGCCTGGCCGGTCAGCTGCCTGCAAGTAGCCGCGGTCTGCCGCAGGCCCATTTTGCACGCGGTGTGTATTTTAGTTATGCAGGCCGCGTTCCGTTTAGCCATCTGATTTATCCGGTGCCGGAACCGGGTGGTCTGGGTATTCATCTGACCCTGGATATGGGCGGCCAGCCGCGCTTTGGTCCGGATGTGGAATGGATTGATACACCTGATTATACCGTGGACCCTGCCCGTGCCGAACGTTTTGCCGCAGCAATTCGTAAATGGTGGCCGGGCCTGGAACCGGAACGCCTGCAGCCTGCATATGCCGGTGTTCGTCCGAAAATTGTTGGTCCGGGCGAAGCCGATGCAGATTTTCAGATTGATGGCCCGGCCGAACATCGCGTTCCGGGTCTGATTAATCTGCTGGGTATTGAAAGTCCGGGCCTGACCGCCGCCCTGGCTATTGGTGAAGAAGTGGCCCGTCGTATTGCAGCACCTCAGGGCTAA(SEQ ID NO.4)
SNU86518.1 nucleotide sequence (codon optimized)
ATGGATAAGGTGGATTGCGTTGTTATTGGCGCAGGTGTTGTTGGCCTGGCAGTGGCACGTACCATGGCAATGGCCGGTCGCGAAGTTGTGGTGCTGGAAAGCGAACGTGCCATTGGTACCGGTACCAGCAGTCGTAATAGTGAAGTGATTCATGGTGGCATTTATTATCCGCCGGGCAGTCGTAAAGCCACCCTGTGCGTTGAAGGTAAACATCGTCTGTATGAATTTTGCGCAAGTCATGGTGTTGAACATCGTCGCTGCGGTAAACTGATTGTTGCCACCACCGATCATCAGGTTGCAGAACTGGAAGCAATTGCAGCCAATGCACGTGCCAGCGGTGTTGATGATCTGCAGTGGCTGAGCGCAGCAGAAGTTGCCCAGCGTGAACCGGCACTGCATACCTTTGGCGCACTGCTGAGCCCGAGCACCGGCATTGTGGATAGCCATGGTCTGATGCTGGCCCTGCAGGGCGATGCCGAAAATGCCGGCGCAATGCTGGCCTTTGAAGCCCGCGTGACCGGTGCACGTGTTGGTCGCGCAGAAGGTATTGAACTGGATGTTGAAACCGAAGGCGTGACCAGCACCCTGCTGGCCAATACCGTTGTGAATAGCGCCGGTCTGCATGCAGTGGATATTGCACGCCGCTTTGATGGCCTGGCCAGTGAACATATTCCGCAGCGCTATTATGCCAAAGGTAGTTATTTTACCTGTGCCCAGCGCGCACCTTTTACCCATCTGATTTATCCGGTTCCGGAACCGGGTGGCCTGGGCGTTCATCTGACCCTGGATCTGGGTGGTCAGGCACGTTTTGGTCCGAATGTTCAGTGGGTGGATGAAATTGATTATACCGTTAATCCGAGTGATGGTGATGGTTTTTATGCCGCCGTTCGCCGTTATTGGCCGACCCTGGCCGATGGCGCACTGCAGCCTGGTTATGCAGGCATTCGTCCGAAAATTAGTGGTCCGGGCGAACCGGCCGCCGATTTTCGTATTGATGGCCCGGCCGTGCATGGTGTTGCAGGCCTGGTGAATCTGTTTGGTATTGAAAGTCCGGGTCTGACCGCCAGCCTGGCAATTGCAGAAGCAGTGCGCGCCGCCCTGAGCTAA(SEQ ID NO.5)
S2DJ52.1 nucleotide sequence (after codon optimization)
ATGGATTTTCAGGTTATCATCATCGGTGGTGGTATTGTTGGTCTGGCCACCGGCCTGAAAATTAAACAGCGTAATCCGAACATTAAAGTGGCGCTGCTGGAAAAAGAAGAAGAAGTTGCAAAACATCAGACCGGTAACAATAGTGGTGTTATTCATAGTGGTCTGTATTATAAACCGGGTAGCCTGAAAGCAAAAAATTGTATTGAAGGTTATCATGAACTGGTTCGTTTTTGTGAAGAAGAAAATATTCCGTTTGAACTGACCGGTAAAGTTGTTGTTGCAACCCGTAAAGAACAGGTTCCGCTGCTGAATAGCCTGCTGGAACGTGGTCTGCAGAATGGTCTGAAAGGTACCCGTAGCATTACCCTGGATGAACTGAAACATTTTGAACCGTATTGTGCAGGTGTTGCAGCAATTCATGTTCCGCAGACCGGTATTGTTGATTATAAACTGGTTGCAGAAAAATATGCAGAAAAATTTCAGATTCTGGGTGGTCAGGTTTTTCTGGGTCATAAAGTTATTAAAGTTGAAACCCAGAATACCGCAAGCATTATTCATACCAGCAAAGGTAGCTTTAGCACCAATCTGCTGATTAATTGTGCAGGTCTGTATAGCGATAAAGTTGCACAGATGAATCAGAAAGAAAGCCTGGATGTTAAAATTATTCCGTTTCGTGGTGAATATTATAAAATTAAAAAAGAACGTGAATATCTGGTTAAAAATCTGATTTATCCGGTTCCGGACCCTAATTTTCCGTTTCTGGGTGTTCATTTTACCCGTATGATGAAAGGTGGTGTTGAAGCAGGTCCGAATGCAGTTCTGGCATTTAAACGTGAAGGTTATAAAAAAAGCCAGGTTAATTTTAGCGAACTGGCAGAAACCCTGAGCTGGCCGGGTTTTCAGAAAGTTGCAAGCAAATATTGGAAAACCGGTATGGGTGAACTGTTTCGTAGCTTTAGCAAAAAAGCATTTACCGATGCACTGAAAGAACTGATTCCGGATATTCAGGAAAGCGATCTGATTGAAGGTGGTGCAGGTGTTCGTGCACAGGCATGTGATCGTACCGGTGGTCTGCTGGATGATTTTTGTATTCGTGAAGATCAGAATGCAATTCATGTTCTGAATGCACCGAGCCCGGCAGCAACCAGCAGCCTGAGCATTGGTGGTACCGTTTGTGAATGGGCACTGAAACGTTTTTAA(SEQ ID NO.6)
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A high catalytic activity L-2-HG dehydrogenase, wherein said high catalytic activity L-2-HG dehydrogenase is derived from any one or more of the group consisting of, but not limited to, vibrio azotemlobus (Azoarcus olearius) BH72, pandora (Pandoraea sputorum) NCTC13161, and bacillus indicus (Indibacter alkaliphilus) LW 1;
specifically, the L-2-HG dehydrogenase is selected from the group consisting of:
(a1) Proteins consisting of the amino acid sequences shown in SEQ ID NO. 1-3;
(a2) A protein having the same or similar function by substituting, deleting and/or adding one or more amino acid residues in the amino acid sequence shown in (a 1);
(a3) A protein having an amino acid sequence composition identical to that of (a 1) or (a 2) of 50% or more and having the same or similar function to that of the protein of (a 1) or (a 2).
2. A polynucleotide capable of encoding the highly catalytically active L-2-HG dehydrogenase of claim 1.
3. The polynucleotide of claim 2, wherein said polynucleotide has the nucleotide sequence of any one of (b 1) - (b 4):
(b1) Nucleotide sequence shown as SEQ ID NO. 4-6;
(b2) A sequence formed by substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence as shown in (b 1);
(b3) A nucleic acid molecule having 50% or more identity to the nucleotide sequence defined in (b 1) or (b 2) and encoding said fusion protein;
(b4) A nucleotide sequence capable of hybridizing to the nucleotide sequence according to any one of (b 1) to (b 3) under stringent conditions and encoding the same functional high catalytic activity L-2-HG dehydrogenase;
further, the polynucleotide is DNA, including cDNA, genomic DNA, or recombinant DNA.
4. A recombinant expression vector comprising at least the polynucleotide of claim 2 or 3;
further, the recombinant expression vector is obtained by operably linking the polynucleotide of claim 2 or 3 to an expression vector comprising a viral vector, a plasmid, a phage, a cosmid, or an artificial chromosome; further, the expression vector is a plasmid, including pACYCDuet-1 plasmid.
5. A host cell comprising the polynucleotide of claim 2 or 3, the recombinant expression vector of claim 4, or capable of expressing the high catalytic activity L-2-HG dehydrogenase of claim 1.
6. Use of a highly catalytically active L-2-HG dehydrogenase according to claim 1, a polynucleotide according to claim 2 or 3, a recombinant expression vector according to claim 4 and/or a host cell according to claim 5 for the preparation of a biosensor for detecting L-2-HG.
7. A biosensor for detecting L-2-HG, wherein the biosensor comprises at least the high catalytic activity L-2-HG dehydrogenase of claim 1, and a redox indicator; wherein the redox reaction indicator is an electron acceptor which can accept the high catalytic activity L-2-HG dehydrogenase to catalyze the L-2-HG to dehydrogenate to generate electrons;
further, the electron acceptor includes azoisophthalol, sodium 3' - [1- [ (anilino) -carbonyl ] -3, 4-tetrazole ] -bis (4-methoxy-6-nitro) benzene-sulfonate, and dichlorophenol indophenol.
8. The biosensor of claim 7, further comprising other reagents, devices and/or apparatus for L-2-HG detection;
further, the reagent comprises a detection buffer;
further, the biosensor for detecting L-2-HG at least comprises: 0.1-50 mu M of resazurin and 0.005-1 mg mL -1 AoL2HGDH and buffers;
the buffer solution is selected from Tris-HCl, HEPES, potassium phosphate, sodium phosphate, MOPS and PBS, the concentration of the buffer solution is 50-100 mM, and the pH value is 5.8-9.0;
further preferably, the biosensor for detecting L-2-HG includes: 67mM PBS (pH 6.6), 1. Mu.M resazurin, 0.05mg mL -1 AoL2HGDH。
9. A detection kit, characterized in that the detection kit comprises the above biosensor for detecting L-2-HG.
10. A method of detecting L-2-HG, the method comprising at least: incubating a sample to be tested with the biosensor according to claim 7 or 8, and analyzing the concentration or presence of L-2-HG;
further, the sample to be detected is a sample containing or suspected of containing L-2-HG, and the sample is a biological sample or an environmental sample, wherein the biological sample comprises a bacterial culture medium, a bacterial lysate, a cell culture medium, a cell lysate, animal serum, animal urine and animal tissue fluid;
still further, the animal is a mammal, including a human.
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