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

An L-2-hydroxyglutarate biosensor based on highly catalytically active L-2-hydroxyglutarate dehydrogenase and its application Download PDF

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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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dehydrogenase
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高超
侯爽
康照琪
马翠卿
许平
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Abstract

本发明属于生物检测技术领域,具体涉及一种基于高催化活性L‑2‑羟基戊二酸脱氢酶的L‑2‑羟基戊二酸生物传感器及其应用。本发明从多种菌株中筛选出具有高催化活性L‑2‑HG脱氢酶,并基于高催化活性L‑2‑HG脱氢酶构建获得L‑2‑HG生物传感器,经试验验证,其响应幅度达2189.25±26.89%,检测下限低至0.042μM,从而可实现对多种细菌、细胞样品以及人体体液中L‑2‑HG的定量检测,从而可应用于L‑2‑HG相关基础研究、L‑2‑HG相关疾病(如肾癌)检测和筛选L‑2‑HG相关药物(如L‑2‑HG激活剂或抑制剂)等诸多领域,因此具有广阔的应用前景。

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

一种基于高催化活性L-2-羟基戊二酸脱氢酶的L-2-羟基戊二 酸生物传感器及其应用A kind of L-2-hydroxyglutarate based on highly catalytically active L-2-hydroxyglutarate dehydrogenase Acid biosensors and their applications

技术领域Technical field

本发明属于生物检测技术领域,具体涉及一种基于高催化活性L-2-羟基戊二酸脱氢酶的L-2-羟基戊二酸生物传感器及其应用。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.

背景技术Background technique

公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。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-羟基戊二酸(L-2-Hydroxyglutarate,L-2-HG)是TCA循环中2-酮基戊二酸(2-ketoglutarate,2-KG)的结构类似物。已有研究表明,L-2-HG在生命体正常生理代谢中发挥多样性的生物学功能,包括参与L-赖氨酸分解与碳饥饿响应、缺氧适应、早期胚胎发育及免疫应答等。在哺乳动物及微生物中,L-2-HG的分解依赖于以FAD作为辅因子的黄素蛋白L-2-HG脱氢酶(在哺乳动物中称为L2HGDH,在微生物中称为LhgO),将其转变为2-KG。L2HGDH突变可导致L-2-HG异常积累,其通过抑制多种2-KG依赖型双加氧酶的活性,导致基因组范围内的组蛋白和DNA甲基化改变、阻止细胞分化,最终引起癌症发生,目前已经在L-2-羟基戊二酸尿症、肾癌、胰腺癌及结直肠癌等多种疾病中发现L-2-HG的积累。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是生命体中一种关键的信号和免疫代谢物,且其可作为L-2-HG相关疾病诊断和预后的关键生物标志物,开发精确、便捷、高通量、低成本的L-2-HG检测方法具有重要科学价值与临床意义。当前L-2-HG的检测主要依赖于液相色谱-串联质谱(LC-MS/MS)、气相色谱-串联质谱(GC-MS/MS)、磁共振波谱(MRS)等技术手段,这些方法均较为昂贵、繁琐、耗时、难以实现L-2-HG与其对映异构体D-2-羟基戊二酸(D-2-hydroxyglutarate,D-2-HG)的分离、需要专业的实验操作人员,且与高通量分析不兼容,限制了L-2-HG相关疾病诊疗及功能多样性研究的开展。另外,近期报道了一种基于L-2-HG特异性转录调控因子与荧光共振能量转移技术的L-2-HG生物传感器LHGFR。优化后传感器LHGFR0N3C与LHGFR0N7C对L-2-HG的最大荧光信号变化(ΔRmax,即响应幅度)分别为56.13±0.29%与60.37±1.30%。另外,LHGFR0N3C与LHGFR0N7C对血清中L-2-HG的检测下限(LOD)分别为5.84μM与1.68μM,对尿液中L-2-HG的检测下限分别为15.74μM与0.92μM。较低的响应幅度与较高的检测下限使得LHGFR在低浓度L-2-HG精准定量方面受到限制,如无法实现健康成人体液中L-2-HG的检测。因此,亟需开发一种高通量、低成本、高响应幅度、低检测下限的高性能L-2-HG生物传感器以满足L-2-HG定量检测的需要。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.

发明内容Contents of the invention

针对上述现有技术的不足,发明人经长期的技术与实践探索,提供一种基于高催化活性L-2-HG脱氢酶的L-2-HG生物传感器及其应用。本发明从多种菌株中筛选出具有高催化活性L-2-HG脱氢酶,并基于高催化活性L-2-HG脱氢酶构建获得L-2-HG生物传感器。经试验验证,所述L-2-HG生物传感器其响应幅度达2189.25±26.89%,检测下限低至0.042μM,从而可实现对多种细菌、细胞样品以及人体体液中L-2-HG的定量检测。基于上述研究成果,从而完成本发明。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.

为实现上述技术目的,本发明采用如下技术方案:In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

本发明的第一个方面,提供一种高催化活性L-2-HG脱氢酶,所述高催化活性L-2-HG脱氢酶来自包括但不限于固氮弧菌(Azoarcus olearius)BH72、潘多拉菌(Pandoraeasputorum)NCTC13161和印度杆菌(Indibacter alkaliphilus)LW1中的任意一种或多种。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.

具体的,所述L-2-HG脱氢酶选自:Specifically, the L-2-HG dehydrogenase is selected from:

(a1)SEQ ID NO.1-3任一项所示氨基酸序列组成的蛋白质;(a1) A protein consisting of the amino acid sequence shown in any one of SEQ ID NO. 1-3;

(a2)将(a1)所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失和/或添加且具有相同或相近功能的蛋白质;(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)与(a1)或(a2)所示的氨基酸序列组成具有同一性达到50%或50%以上并且具有与(a1)或(a2)所示的蛋白质具有相同或相近功能的蛋白质。(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).

本发明的第二个方面,提供一种多聚核苷酸,所述多聚核苷酸能够编码上述高催化活性L-2-HG脱氢酶。A second aspect of the present invention provides a polynucleotide capable of encoding the above-mentioned high catalytic activity L-2-HG dehydrogenase.

具体的,所述多聚核苷酸具有(b1)-(b4)中任一所述核苷酸序列:Specifically, the polynucleotide has any one of the nucleotide sequences (b1)-(b4):

(b1)如SEQ ID NO.4-6所示的核苷酸序列;(b1) The nucleotide sequence shown in SEQ ID NO.4-6;

(b2)如(b1)所示的核苷酸序列经过一个或多个核苷酸的取代、缺失和/或添加形成的序列;(b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1);

(b3)与(b1)或(b2)限定的核苷酸序列具有50%或50%以上的同一性,且编码所述融合蛋白的核酸分子;(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)在严格条件下能够与如(b1)-(b3)中任一项所述核苷酸序列杂交并编码相同功能高催化活性L-2-HG脱氢酶的核苷酸序列。(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.

本发明的第三个方面,提供一种重组表达载体,所述重组表达载体至少包含上述多聚核苷酸。A third aspect of the present invention provides a recombinant expression vector, which at least contains the above polynucleotide.

本发明的第四个方面,提供一种宿主细胞,所述宿主细胞含有上述多聚核苷酸、上述重组表达载体或者能够表达上述高催化活性L-2-HG脱氢酶。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.

本发明的第五个方面,提供上述高催化活性L-2-HG脱氢酶、多聚核苷酸、重组表达载体和/或细胞在制备检测L-2-HG的生物传感器中的应用。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.

本发明的第六个方面,提供一种检测L-2-HG的生物传感器(可将其命名为EaLHGFR),所述生物传感器至少包含上述高催化活性L-2-HG脱氢酶,以及氧化还原反应指示剂;其中,所述氧化还原反应指示剂可以为可接受L-2-HG脱氢酶催化L-2-HG脱氢产生电子的电子受体。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.

上述生物传感器在实际应用过程中,可以以检测试剂盒形式存在。In actual application, the above-mentioned biosensor can exist in the form of a detection kit.

本发明的第七个方面,提供一种检测L-2-HG的方法,所述方法至少包括:将待测样品与所述生物传感器进行孵育,分析L-2-HG的浓度或有无。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.

与现有技术方案相比,上述一个或多个技术方案具有如下有益技术效果:Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial technical effects:

(1)上述技术方案基于序列比对分析,从不同菌株中筛选出多个具有高催化活性的L-2-HG脱氢酶;其中,在A.olearius BH72中鉴定得到一种具有高底物特异性及高催化活性的L-2-HG脱氢酶AoL2HGDH;AoL2HGDH以FAD为辅因子,催化L-2-HG转化为2-KG的同时可将电子直接传递给氧化还原反应指示剂,无需额外电子传递介体;(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)上述技术方案开发的基于高催化活性L-2-HG脱氢酶的L-2-HG生物传感器EaLHGFR,具有响应幅度高、检测下限低的特点,且检测体系仅包含AoL2HGDH与氧化还原反应指示剂两组分,制备简易、成分简单、成本较低、易于操作、可实现高通量检测;(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)上述技术方案开发的基于高催化活性L-2-HG脱氢酶的L-2-HG生物传感器EaLHGFR,适用于不同细菌、细胞及人体血清、尿液等生物样品中L-2-HG的定量检测,检测结果与当前L-2-HG标准检测方法LC-MS/MS高度一致,在各种生物样品的L-2-HG检测中具有广阔的应用前景。(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.

附图说明Description of the drawings

构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。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.

图1为本发明实施例1中不同来源L-2-HG脱氢酶的酶活比较。Figure 1 is a comparison of the enzyme activities of L-2-HG dehydrogenases from different sources in Example 1 of the present invention.

图2为本发明实施例1中AoL2HGDH表达纯化的SDS-PAGE验证。Figure 2 is SDS-PAGE verification of expression and purification of AoL2HGDH in Example 1 of the present invention.

图3为本发明实施例1中AoL2HGDH对L-2-HG的动力学参数测定及底物特异性分析;其中,A为AoL2HGDH对L-2-HG的动力学参数测定,B为AoL2HGDH的底物特异性分析。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.

图4为本发明实施例2中基于AoL2HGDH的L-2-HG生物传感器检测原理示意图。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.

图5为本发明实施例2中初始L-2-HG生物传感器EaLHGFR-1对L-2-HG的剂量-响应曲线。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.

图6为本发明实施例2中L-2-HG生物传感器的系统优化。Figure 6 shows the system optimization of the L-2-HG biosensor in Example 2 of the present invention.

图7为本发明实施例2中优化后L-2-HG生物传感器EaLHGFR-2对L-2-HG的剂量-响应曲线。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.

图8为本发明实施例3中EaLHGFR-2与LC-MS/MS用于细菌样品L-2-HG检测的一致性分析;其中,A为EaLHGFR-2与LC-MS/MS用于细菌裂解物中L-2-HG检测的一致性分析,B为EaLHGFR-2与LC-MS/MS用于细菌培养基中L-2-HG检测的一致性分析。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.

图9为本发明实施例3中应用EaLHGFR-2实现细菌胞内外L-2-HG的定量检测;其中,A为细菌胞内L-2-HG定量检测的结果,B为细菌胞外L-2-HG定量检测的结果。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.

图10为本发明实施例4中EaLHGFR-2与LC-MS/MS用于细胞样品L-2-HG检测的一致性分析;其中,A为EaLHGFR-2与LC-MS/MS用于细胞裂解物中L-2-HG检测的一致性分析,B为EaLHGFR-2与LC-MS/MS用于细胞培养基中L-2-HG检测的一致性分析。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.

图11为本发明实施例4中应用EaLHGFR-2实现细胞胞内外L-2-HG的定量检测;其中,A为细胞胞内L-2-HG定量检测的结果,B为细胞胞外L-2-HG定量检测的结果。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.

图12为本发明实施例5中EaLHGFR-2与LC-MS/MS用于人体体液L-2-HG检测的一致性分析;其中,A为EaLHGFR-2与LC-MS/MS用于人体血清中L-2-HG检测的一致性分析,B为EaLHGFR-2与LC-MS/MS用于人体尿液中L-2-HG检测的一致性分析。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.

图13为本发明实施例5中应用EaLHGFR-2实现人体体液中L-2-HG的定量检测;其中,A为人体血清L-2-HG定量检测的结果,B为人体尿液L-2-HG定量检测的结果。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.

具体实施方式Detailed ways

应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。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.

需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。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.

本发明的一个典型具体实施方式中,提供一种高催化活性L-2-HG脱氢酶,所述高催化活性L-2-HG脱氢酶来自包括但不限于固氮弧菌(Azoarcus olearius)BH72、潘多拉菌(Pandoraea sputorum)NCTC13161和印度杆菌(Indibacter alkaliphilus)LW1中的任意一种或多种;进一步来源于固氮弧菌(Azoarcus olearius)BH72,此时将该高催化活性L-2-HG脱氢酶命名为AoL2HGDH,其GeneBank号为CAL94536.1;AoL2HGDH共价结合FAD作为辅因子,催化L-2-HG生成2-KG,对L-2-HG具有高底物特异性及高脱氢活性。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.

所述L-2-HG脱氢酶选自:The L-2-HG dehydrogenase is selected from:

(a1)SEQ ID NO.1-3所示氨基酸序列组成的蛋白质;(a1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1-3;

(a2)将(a1)所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失和/或添加且具有相同或相近功能的蛋白质;(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)与(a1)或(a2)所示的氨基酸序列组成具有同一性达到50%或50%以上并且具有与(a1)或(a2)所示的蛋白质具有相同或相近功能的蛋白质。(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).

其中,所述(a2)中,所述一个或多个氨基酸残基的取代、缺失和/或添加一般为不超过15个氨基酸残基的取代和/或缺失和/或添加。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.

上述(a1)–(a3)中所示蛋白质可以人工合成,也可先合成其编码基因,再进行生物表达获得。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.

本发明一个或多个具体实施方式中,提供一种多聚核苷酸,所述多聚核苷酸能够编码上述高催化活性L-2-HG脱氢酶。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.

具体的,所述多聚核苷酸具有(b1)-(b4)中任一所述核苷酸序列:Specifically, the polynucleotide has any one of the nucleotide sequences (b1)-(b4):

(b1)如SEQ ID NO.4-6所示的核苷酸序列;(b1) The nucleotide sequence shown in SEQ ID NO.4-6;

(b2)如(b1)所示的核苷酸序列经过一个或多个核苷酸的取代、缺失和/或添加形成的序列;(b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1);

(b3)与(b1)或(b2)限定的核苷酸序列具有50%或50%以上的同一性,且编码所述融合蛋白的核酸分子;(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)在严格条件下能够与如(b1)-(b3)中任一项所述核苷酸序列杂交并编码相同功能高催化活性L-2-HG脱氢酶的核苷酸序列。(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.

需要说明的是,术语“同一性”指与氨基酸/核苷酸序列的序列相似性。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。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.

上述50%以上同一性,可以为50%、60%、70%、80%、85%、90%、95%或99%以上的同一性。The above-mentioned 50% or more identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% or more identity.

所述多聚核苷酸可以是DNA,如cDNA、基因组DNA或重组DNA等,在此不做具体限定。The polynucleotide may be DNA, such as cDNA, genomic DNA, or recombinant DNA, and is not specifically limited here.

本发明一个或多个具体实施方式中,提供一种重组表达载体,所述重组表达载体至少包含上述多聚核苷酸。In one or more specific embodiments of the present invention, a recombinant expression vector is provided, which at least contains the above polynucleotide.

所述重组表达载体通过上述多聚核苷酸有效地连接到表达载体上获得,所述表达载体包括病毒载体(包括腺病毒载体、逆转录病毒载体或腺伴随病毒载体)、质粒、噬菌体、黏粒或人工染色体;进一步的,所述表达载体可以为质粒,如pACYCDuet-1质粒。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.

本发明一个或多个具体实施方式中,提供一种宿主细胞,所述宿主细胞含有上述多聚核苷酸、上述重组表达载体或者能够表达上述高催化活性L-2-HG脱氢酶。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.

所述宿主细胞包括细菌细胞或真菌细胞;The host cells include bacterial cells or fungal cells;

所述细菌可以为埃希氏菌属、农杆菌属、芽孢杆菌属、链霉菌属、假单胞菌属或葡萄球菌属中的任意一种或多种。The bacteria may be any one or more of the genus Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas or Staphylococcus.

本发明的一个或多个具体实施方式中,所述细菌为大肠杆菌(如BL21(DE3))、根癌农杆菌(如GV3101)、发根农杆菌、乳酸乳球菌、枯草芽孢杆菌、蜡状芽孢杆菌或荧光假单胞菌。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.

本发明一个或多个具体实施方式中,提供上述高催化活性L-2-HG脱氢酶、多聚核苷酸、重组表达载体和/或细胞在制备检测L-2-HG的生物传感器中的应用。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.

本发明一个或多个具体实施方式中,提供一种检测L-2-HG的生物传感器(可将其命名为EaLHGFR),所述生物传感器至少包含上述高催化活性L-2-HG脱氢酶,以及氧化还原反应指示剂;其中,所述氧化还原反应指示剂可以为可接受L-2-HG脱氢酶催化L-2-HG脱氢产生电子的电子受体。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.

所述电子受体包括但不限于天青、3’-[1-[(苯胺基)-羰基]-3,4-四唑]-双(4-甲氧基-6-硝基)苯-磺酸钠和二氯酚靛酚。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.

所述生物传感器还可以包括其他用于L-2-HG检测的试剂、装置和/或设备;The biosensor may also include other reagents, devices and/or equipment for L-2-HG detection;

所述试剂包括检测缓冲液。The reagents include detection buffer.

本发明的又一具体实施方式中,所述检测L-2-HG的生物传感器包括:0.1~50μM刃天青、0.005~1mg mL-1AoL2HGDH,以及缓冲液;所述缓冲液选自Tris-HCl、HEPES、磷酸钾、磷酸钠、MOPS以及PBS,所述缓冲液浓度为50~100mM,pH为5.8~9.0;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;

进一步优选的,所述检测L-2-HG的生物传感器包括:67mM PBS(pH 6.6)、1μM刃天青、0.05mg mL-1AoL2HGDH。在该条件下,当反应体系中出现L-2-HG时,AoL2HGDH催化L-2-HG脱氢,伴随着共价结合的FAD转变为FADH2,FADH2进一步将电子传递至刃天青,生成高红移荧光分子试卤灵(发射峰位于587nm),且此时,其对0.5μM和1μM L-2-HG具有最高的响应幅度,经试验验证,其响应幅度为2189.25±26.89%、检测下限为0.042μM,从而实现对L-2-HG的高灵敏度检测。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.

上述生物传感器在实际应用过程中,可以以检测试剂盒形式存在。In actual application, the above-mentioned biosensor can exist in the form of a detection kit.

试剂盒可包含实施本发明方法所用的材料或试剂(包括高催化活性L-2-HG脱氢酶、氧化还原反应指示剂等)。试剂盒可以包括储存反应试剂(例如在合适容器中的L-2-HG脱氢酶、氧化还原反应指示剂等)和/或支持材料(例如缓冲液、实施检测的说明书等)。例如,试剂盒可以包括一个或多个含有相应反应试剂和/或支持材料的容器(例如盒子)。这样的内容物可一起或分开递送给既定的接受者。例如,第一个容器可含有用于测定的酶,第二个容器含有氧化还原反应指示剂、而第三个容器含有缓冲液。所述试剂盒还可含有适合容纳所述试剂或容器的隔室。在此不做具体限定。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.

本发明一个或多个具体实施方式中,提供一种检测L-2-HG的方法,所述方法至少包括:将待测样品与所述生物传感器进行孵育,分析L-2-HG的浓度或有无。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.

其中,所述待测样品为含有或疑似含有L-2-HG的样品,所述样品可以为生物样品或环境样品,所述生物样品包括但不限于细菌培养基、细菌裂解液、细胞培养基、细胞裂解液、动物血清、动物尿液和动物组织液;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;

其中,所述动物可以为哺乳动物,其中,人是优选的。Wherein, the animal may be a mammal, among which humans are preferred.

通过上述方法对L-2-HG进行定性或定量检测,从而可应用于L-2-HG相关基础研究、L-2-HG相关疾病(如肾癌)检测和筛选L-2-HG相关药物(如L-2-HG激活剂或抑制剂)等诸多领域,因此具有广阔的应用前景。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.

以下结合具体实例对本发明作进一步的说明,以下实例仅是为了解释本发明,并不对其内容进行限定。所使用的实验方法,未做具体说明的,均为常规方法。所使用的菌株、细胞、材料、试剂等,如无特殊说明,均为从商业途径获得。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.

实施例1:高催化活性L-2-HG脱氢酶AoL2HGDH蛋白的鉴定与表征Example 1: Identification and characterization of highly catalytically active L-2-HG dehydrogenase AoL2HGDH protein

(1)不同L-2-HG脱氢酶同源蛋白的外源表达及酶活比较(1) Comparison of exogenous expression and enzyme activities of different L-2-HG dehydrogenase homologous proteins

基于序列比对分析,获得十种细菌、真菌及植物来源的L-2-HG脱氢酶同源蛋白核苷酸序列,委托通用生物系统(安徽)有限公司合成,连接至pACYCDuet-1质粒后保存于E.coli BL21(DE3)菌株;将表达菌株分别接入含氯霉素(40μg mL-1)的LB液体培养基,在37℃与180rpm的条件下培养至OD600nm约0.6,加入1mM IPTG,在23℃与160rpm的条件下过夜诱导蛋白表达;离心收集菌体,高压破碎后获得各L-2-HG脱氢酶的粗酶液;各L-2-HG脱氢酶对L-2-HG活性测定在800μL反应体系中进行,包含50mM Tris-HCl(pH 7.4)、100μM DCPIP、100μM L-2-HG及40μL粗酶液,通过在30℃下检测600nm处吸光度的变化确定各L-2-HG脱氢酶对L-2-HG的催化活性。结果如附图1所示,其中来源于A.olearius BH72的L-2-HG脱氢酶(GeneBank:CAL94536.1)具备对L-2-HG最高的催化活性,将其命名为AoL2HGDH,其氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.4所示。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)AoL2HGDH的表达纯化(2) Expression and purification of AoL2HGDH

将携带AoL2HGDH表达载体的E.coli BL21(DE3)菌株在LB液体培养基中活化两代后,接种至含氯霉素(40μg mL-1)的500mL LB液体培养基中,在37℃与180rpm的条件下培养至OD600nm约0.6,加入1mM IPTG,在23℃与160rpm的条件下过夜诱导AoL2HGDH表达;离心收集菌体,使用结合缓冲液洗涤两次并重悬至OD600nm为20,加入1mM PMSF与10%甘油,高压破碎菌体,12,000rpm、4℃离心50min去除细胞碎片后获得AoL2HGDH的粗酶液;所得粗酶液经0.22μm的滤头过滤后,使用5mL镍柱分离纯化,采用不同浓度的洗脱缓冲液洗脱获得纯化的AoL2HGDH;使用SDS-PAGE检测AoL2HGDH的纯度,结果如附图2所示。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)AoL2HGDH的动力学参数测定及底物特异性分析(3) Determination of kinetic parameters and substrate specificity analysis of AoL2HGDH

AoL2HGDH对L-2-HG的动力学参数测定在800μL反应体系中进行,包含50mM Tris-HCl(pH 7.4)、100μM DCPIP、梯度浓度L-2-HG及适量纯化后的AoL2HGDH蛋白,通过在30℃下检测600nm处吸光度的变化确定AoL2HGDH对梯度浓度L-2-HG的催化活性,拟合计算AoL2HGDH对L-2-HG的动力学参数;结果如附图3A所示,AoL2HGDH对L-2-HG的Vmax值为50.85±1.66U mg-1、Km值为29.12±2.52μM。AoL2HGDH的底物特异性分析反应体系同上,仅将L-2-HG替换为10μM D-2-HG、L-酒石酸、D-酒石酸、L-甘油酸、D-甘油酸、L-乳酸、D-乳酸等L-2-HG结构类似物,通过测定AoL2HGDH对不同结构类似物的活性确定其底物特异性;结果如附图3B所示,AoL2HGDH具备对L-2-HG的高度特异性。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.

其中,上述步骤(1)~(2)中所述的LB培养基配方为:酵母粉5g L-1;蛋白胨10g L-1;NaCl 10g L-1,pH 7.0;121℃灭菌20min。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.

上述步骤(2)中所述的结合缓冲液配方为:20mM Na2HPO4,20mM咪唑,500mM NaCl,调整pH为7.4;洗脱缓冲液配方为:20mM Na2HPO4,500mM咪唑,500mM NaCl,调整pH为7.4。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.

实施例2:基于高催化活性L-2-HG脱氢酶AoL2HGDH的L-2-HG生物传感器的构建与优化Example 2: Construction and optimization of L-2-HG biosensor based on highly catalytically active L-2-HG dehydrogenase AoL2HGDH

(1)基于高催化活性L-2-HG脱氢酶AoL2HGDH的L-2-HG生物传感器的构建原理(1) Construction principle of L-2-HG biosensor based on highly catalytic activity L-2-HG dehydrogenase AoL2HGDH

AoL2HGDH具备高催化活性及高底物特异性,可作为理想的催化元件与刃天青介导的氧化还原报告系统相结合,开发一种基于酶法的L-2-HG生物传感器(EaLHGFR)。EaLHGFR检测原理如附图4所示,其中AoL2HGDH催化L-2-HG脱氢生成2-KG,同时将共价结合的FAD还原为FADH2;产生的FADH2可进一步将刃天青还原生成高红移荧光分子试卤灵。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)初始L-2-HG生物传感器EaLHGFR-1的构建与性质表征(2) Construction and characterization of the initial L-2-HG biosensor EaLHGFR-1

配制由50mM Tris-HCl(pH 7.4)、15μM刃天青、0.1mg mL-1AoL2HGDH构成的L-2-HG生物传感器工作液(初始L-2-HG生物传感器命名为EaLHGFR-1),配制梯度浓度的L-2-HG溶液(0mM至20mM),取75μLEaLHGFR-1工作液与25μL L-2-HG溶液于黑色96孔板中混合,于避光条件下30℃反应60min,使用荧光酶标仪测定在544nm的激发波长下,590nm处的荧光发射强度。以L-2-HG的浓度为横坐标,以EaLHGFR-1荧光强度为纵坐标,绘制EaLHGFR-1对L-2-HG的剂量-响应曲线。结果如附图5所示,EaLHGFR-1以剂量依赖的方式响应于L-2-HG的添加,其响应幅度(即最大荧光强度变化,ΔFmax)为1933.73±38.99%,检测下限为0.976μM。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)L-2-HG生物传感器的系统优化(3) System optimization of L-2-HG biosensor

为提高L-2-HG生物传感器的检测灵敏性,以单因素筛选的策略针对检测缓冲液类型(50mM Tris-HCl[pH 7.4]、100mM Tris-HCl[pH 7.4]、100mM HEPES[pH 7.4]、100mM磷酸钾[pH 7.4]、100mM磷酸钠[pH 7.4]、100mM MOPS[pH 7.4]及67mM PBS[pH 7.4])、检测缓冲液pH(5.8、6.2、6.6、7.0、7.4、7.8、8.2、8.6、9.0)、刃天青浓度(0.1、0.5、1、5、10、15、50μM)及AoL2HGDH浓度(0.005、0.01、0.05、0.07、0.1、0.5、1mg mL-1)进行系统优化,以生物传感器对低浓度L-2-HG(0.5μM及1μM)的响应幅度确定生物传感器的检测灵敏度。结果如附图6所示,由67mM PBS(pH 6.6)、1μM刃天青、0.05mg mL-1AoL2HGDH构成的生物传感器变体具备对0.5μM和1μM L-2-HG最高的响应幅度,分别由EaLHGFR-1的29%和66%提高到488%和885%,将该传感器变体命名为EaLHGFR-2。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)优化后L-2-HG生物传感器EaLHGFR-2的性质表征(4) Characterization of the optimized L-2-HG biosensor EaLHGFR-2

以(2)中所述方法测定EaLHGFR-2对L-2-HG的剂量-响应曲线。结果如附图7所示,EaLHGFR-2以剂量依赖的方式响应于L-2-HG的添加,其响应幅度达2189.25±26.89%,检测下限低至0.042μM。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.

实施例3:EaLHGFR-2在细菌胞内、胞外L-2-HG定量检测中的应用Example 3: Application of EaLHGFR-2 in quantitative detection of bacterial intracellular and extracellular L-2-HG

(1)细菌培养基和细菌裂解液的制备(1) Preparation of bacterial culture medium and bacterial lysate

将P.putida KT2440菌株在含5g L-1葡萄糖为唯一碳源的无机盐培养基中培养至对数中期;收集2mL培养物,于12,000rpm、4℃离心10min,收集上清液,作为细菌培养基样品;收集15mL培养物,于6,000rpm、4℃离心10min收集菌体,使用1mL乙醇重悬后于90℃抽提胞内代谢物,于真空离心机彻底干燥后使用适量蒸馏水重悬,作为细菌裂解液样品。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)EaLHGFR-2与LC-MS/MS的检测一致性分析(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

在上述制备得到的细菌培养基与细菌裂解液中加入梯度浓度L-2-HG(1、3、5、10、30、50、70μM),与EaLHGFR-2工作液以1:3体积比于黑色96孔板中混合,于避光条件下30℃反应60min后使用荧光酶标仪测定在544nm的激发波长下,590nm处的荧光发射强度;将所得荧光强度代入实施例2中所述EaLHGFR-2对L-2-HG的剂量-响应曲线中计算得到上述样品具体L-2-HG浓度。另取上述相同样品,使用Thermo Ultimate 3000快速分离液相色谱系统(美国ThermoFisher Scientific公司)串联Bruker impact HD ESI-Q-TOF质谱仪(美国BrukerDaltonics公司)搭配Chirobiotic R色谱柱(250×4.6mm)(美国Supelco Analytical公司)确定上述样品中L-2-HG的实际浓度。EaLHGFR-2与LC-MS/MS对细菌胞内、胞外L-2-HG检测结果的比较如附图8所示,EaLHGFR-2的测定结果与当前标准L-2-HG检测方法LC-MS/MS高度一致,表明其应用于细菌胞内、胞外L-2-HG定量检测的准确性。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)EaLHGFR-2在细菌胞内、胞外L-2-HG定量检测中的应用(3) Application of EaLHGFR-2 in quantitative detection of bacterial intracellular and extracellular L-2-HG

将P.putida KT2440和P.putida KT2440(ΔlhgO)在含5g L-1葡萄糖或4g L-1葡萄糖+1g L-1戊二酸为碳源的无机盐培养基中培养至对数中期,使用与(1)中相同方法制备细菌裂解液和细菌培养基样品,按照(2)中方法使用EaLHGFR-2测定不同样品中L-2-HG的具体浓度。结果如附图9所示,lhgO敲除菌在以葡萄糖+戊二酸为碳源培养时,胞内和胞外L-2-HG水平显著升高,证明EaLHGFR-2适用于细菌胞内、胞外L-2-HG的定量检测。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.

其中,上述步骤(1)、(3)中所述不含碳源的无机盐培养基(1L)配方为:1g NH4Cl,2.26g KH2PO4,4.1g K2HPO4,2.24g NaH2PO4·H2O,3.34g Na2HPO4,10mL金属离子混合液,NaOH调pH至7.0,121℃灭菌20分钟。金属离子混合液(1L):14.8g MgSO4·7H2O,550mgFeSO4·7H2O,45mg MnSO4·4H2O,200μL H2SO4Among 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 .

实施例4:EaLHGFR-2在细胞胞内、胞外L-2-HG定量检测中的应用Example 4: Application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG

(1)细菌培养基和细菌裂解液的制备(1) Preparation of bacterial culture medium and bacterial lysate

将人胚胎肾细胞(HEK293FT)在含10%胎牛血清、1%青霉素-链霉素的高糖DMEM培养基中,于37℃、5% CO2的条件下静置培养24h;收集2mL培养物,与甲醇按照1:1的体积比混合后于12,000rpm、4℃离心10min,收集上清液,作为细胞培养基样品;收集贴壁培养的细胞,加入适量提取液(甲醇:乙腈:蒸馏水=5:3:1;每1×106个细胞加入500μL提取液),以反复冻融法破碎细胞,抽提胞内代谢物,于真空离心机彻底干燥后使用适量蒸馏水重悬,作为细胞裂解液样品。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)EaLHGFR-2与LC-MS/MS的检测一致性分析(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

在上述制备得到的细胞培养基与细胞裂解液中加入梯度浓度L-2-HG(1、3、5、10、30、50、70μM),与EaLHGFR-2工作液以1:3体积比于黑色96孔板中混合,于避光条件下30℃反应60min后使用荧光酶标仪测定在544nm的激发波长下,590nm处的荧光发射强度;将所得荧光强度代入实施例2中所述EaLHGFR-2对L-2-HG的剂量-响应曲线中计算得到上述样品具体L-2-HG浓度。另取上述相同样品,以实施例3中所述方法使用LC-MS/MS分析确定上述样品中L-2-HG的实际浓度。EaLHGFR-2与LC-MS/MS对细胞胞内、胞外L-2-HG检测结果的比较如附图10所示,EaLHGFR-2的测定结果与当前标准L-2-HG检测方法LC-MS/MS高度一致,表明其应用于细胞胞内、胞外L-2-HG定量检测的准确性。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)EaLHGFR-2在细胞胞内、胞外L-2-HG定量检测中的应用(3) Application of EaLHGFR-2 in quantitative detection of intracellular and extracellular L-2-HG

将HEK293FT细胞在21% O2条件下静置培养24h,之后转移至ProOx C21精密氧气控制系统中,以0.5% O2的条件继续培养24h,使用与(1)中相同方法分别制备HEK293FT在常氧与缺氧条件培养下的细胞裂解液和细胞培养基样品,按照(2)中方法使用EaLHGFR-2测定不同样品中L-2-HG的具体浓度。结果如附图11所示,缺氧处理导致HEK293FT细胞胞内和胞外L-2-HG水平分别提高至2.59倍与1.53倍,证明EaLHGFR-2适用于细胞胞内、胞外L-2-HG的定量检测。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.

实施例5:EaLHGFR-2在人体血清、尿液L-2-HG定量检测中的应用Example 5: Application of EaLHGFR-2 in quantitative detection of L-2-HG in human serum and urine

(1)人体血清和尿液样品制备(1) Preparation of human serum and urine samples

按照伦理规范,通过静脉采血法收集健康成年人血液于促凝管中,室温放置2h后,在4℃、3,000rpm离心10min,上清经0.22μm滤膜过滤后获得血清,储存于-20℃备用。直接采集健康成年人尿液,经0.22μm滤膜过滤后,储存于-20℃备用。应用EaLHGFR-2定量检测人体血清、尿液中L-2-HG浓度须对血清、尿液除蛋白处理,即采用商品化的基于高氯酸的去蛋白试剂盒,按照试剂盒步骤进行去蛋白操作。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)EaLHGFR-2与LC-MS/MS的检测一致性分析(2) Consistency analysis of EaLHGFR-2 and LC-MS/MS detection

在上述除蛋白得到的血清和尿液样品中加入梯度浓度L-2-HG(1、3、5、10、30、50、70μM),与EaLHGFR-2工作液以1:3体积比于黑色96孔板中混合,于避光条件下30℃反应60min后使用荧光酶标仪测定在544nm的激发波长下,590nm处的荧光发射强度;将所得荧光强度代入实施例2中所述EaLHGFR-2对L-2-HG的剂量-响应曲线中计算得到上述样品具体L-2-HG浓度。另取上述相同样品,以实施例3中所述方法使用LC-MS/MS分析确定上述样品中L-2-HG的实际浓度。EaLHGFR-2与LC-MS/MS对人体血清、尿液中L-2-HG检测结果的比较如附图12所示,EaLHGFR-2的测定结果与当前标准L-2-HG检测方法LC-MS/MS高度一致,表明其应用于人体体液L-2-HG定量检测的准确性。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)EaLHGFR-2在健康人与肾癌患者体液L-2-HG定量检测中的应用(3) Application of EaLHGFR-2 in the quantitative detection of L-2-HG in body fluids of healthy people and renal cancer patients

以(1)中所述方法采集7例健康成人的血清、尿液样品以及4例肾癌患者的血清、尿液样品;按照(2)中方法使用EaLHGFR-2测定健康人与肾癌患者体液内L-2-HG浓度。结果如附图13所示,健康人与肾癌患者血清L-2-HG为0.44μM至0.92μM,且两类人群间血清L-2-HG浓度无显著性差异;另外,健康人尿液中L-2-HG的平均浓度为13.398μM,而肾癌患者尿液中L-2-HG的平均浓度为42.19μM,证明尿液L-2-HG浓度可作为肾癌的生物标志物。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.

本发明中所涉及的核苷酸/氨基酸序列Nucleotide/amino acid sequences involved in the present invention

CAL94536.1(AoL2HGDH)的氨基酸序列Amino acid sequence of CAL94536.1(AoL2HGDH)

METVDCVVVGAGVVGLACARAIAASGREVLILERERAFGTGISSRNSEVIHAGLYYPPGSLKARLSVAGGRMLYAYCESRGVAHRRCGKLVVAAQREALPALARIRARALANGVEELLWLEPGAVKEIEPALDSCGALFSPATGIVDSHGLMLALLGDAERHGATLVLDTPVLGGRAEAGGIVLQTGGEAPMTLQARCVVNAAGLDAVRLAGQLPASSRGLPQAHFARGVYFSYAGRVPFSHLIYPVPEPGGLGIHLTLDMGGQPRFGPDVEWIDTPDYTVDPARAERFAAAIRKWWPGLEPERLQPAYAGVRPKIVGPGEADADFQIDGPAEHRVPGLINLLGIESPGLTAALAIGEEVARRIAAPQG(SEQID NO.1)METVDCVVVGAGVVGLACARAIAASGREVLILERERAFGTGISSRNSEVIHAGLYYPPGSLKARLSVAGGRMLYAYCESRGVAHRRCGKLVVAAQREALPALARIRARALANGVEELLWLEPGAVKEIEPALDSCGALFSPATGIVDSGHGLMLALLGDAERHGATLVLDTPVLGGRAEAGGIVLQTGGEAPMTLQARCVVNAAGLDAVRLAGQLPASSRGLPQAHFAR GVYFSYAGRVPFSHLIYPVPEPGGLGIHLTLDMGGQPRFGPDVEWIDTPDYTVDPARAERFAAAIRKWWPGLEPERLQPAYAGVRPKIVGPGEADADFQIDGPAEHRVPGLINLLGIESPGLTAALAIGEEVARRIAAPQG(SEQID NO.1)

SNU86518.1的氨基酸序列Amino acid sequence of SNU86518.1

MDKVDCVVIGAGVVGLAVARTMAMAGREVVVLESERAIGTGTSSRNSEVIHGGIYYPPGSRKATLCVEGKHRLYEFCASHGVEHRRCGKLIVATTDHQVAELEAIAANARASGVDDLQWLSAAEVAQREPALHTFGALLSPSTGIVDSHGLMLALQGDAENAGAMLAFEARVTGARVGRAEGIELDVETEGVTSTLLANTVVNSAGLHAVDIARRFDGLASEHIPQRYYAKGSYFTCAQRAPFTHLIYPVPEPGGLGVHLTLDLGGQARFGPNVQWVDEIDYTVNPSDGDGFYAAVRRYWPTLADGALQPGYAGIRPKISGPGEPAADFRIDGPAVHGVAGLVNLFGIESPGLTASLAIAEAVRAALS(SEQID NO.2)MDKVDCVVIGAGVVGLAVARTMAMAGREVVVLESERAIGTGTSSRNSEVIHGGIYYPPGSRKATLCVEGKHRLYEFCASHGVEHRRCGKLIVATTDHQVAELEAIAANARASGVDDLQWLSAAAEVAQREPALHTFGALLSPSTGIVDSGHLMLALQGDAENAGAMLAFEARVTGARVGRAEGIELDVETEGVTSTLLANTVVNSAGLHAVDIARRF DGLASEHIPQRYYAKGSYFTCAQRAPFTHLIYPVPEPGLGVHLTLDLGGQARFGPNVQWVDEIDYTVNPSDGDGFYAAVRRYWPTLADGALQPGYAGIRPKISGPGEPAADFRIDGPAVHGVAGLVNLFGIESPGLTASLAIAEAVRAALS(SEQID NO.2)

S2DJ52.1氨基酸序列S2DJ52.1 amino acid sequence

MDFQVIIIGGGIVGLATGLKIKQRNPNIKVALLEKEEEVAKHQTGNNSGVIHSGLYYKPGSLKAKNCIEGYHELVRFCEEENIPFELTGKVVVATRKEQVPLLNSLLERGLQNGLKGTRSITLDELKHFEPYCAGVAAIHVPQTGIVDYKLVAEKYAEKFQILGGQVFLGHKVIKVETQNTASIIHTSKGSFSTNLLINCAGLYSDKVAQMNQKESLDVKIIPFRGEYYKIKKEREYLVKNLIYPVPDPNFPFLGVHFTRMMKGGVEAGPNAVLAFKREGYKKSQVNFSELAETLSWPGFQKVASKYWKTGMGELFRSFSKKAFTDALKELIPDIQESDLIEGGAGVRAQACDRTGGLLDDFCIREDQNAIHVLNAPSPAATSSLSIGGTVCEWALKRF(SEQ ID NO.3)MDFQVIIIGGGIVGLATGLKIKQRNPNIKVALLEKEEEVAKHQTGNNSGVIHSGLYYKPGSLKAKNCIEGYHELVRFCEEENIPFELTGKVVVATRKEQVPLLNSLLERGLQNGLKGTRSITLDELKHFEPYCAGVAAIHVPQTGIVDYKLVAEKYAEKFQILGGQVFLGHKVIKVETQNTASIIHTSKGSSFSTNLLINCAGLYSDKVA QMNQKESLDVKIIPFRGEYYKIKKEREYLVKNLIYPVPDPNFPFLGVHFTRMMKGGVEAGPNAVLAFKREGYKKSQVNFSELAETLSWPGFQKVASKYWKTGMGELFRSFSKKAFTDALKELIPDIQESDLIEGGAGVRAQACDRTGGLLDDFCIREDQNAIHVLNAPSPAATSSLSIGGTVCEWALKRF(SEQ ID NO.3)

CAL94536.1(AoL2HGDH)的核苷酸序列(密码子优化后)Nucleotide sequence of CAL94536.1(AoL2HGDH) (after codon optimization)

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

SNU86518.1核苷酸序列(密码子优化后)SNU86518.1 nucleotide sequence (after codon optimization)

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

S2DJ52.1核苷酸序列(密码子优化后)S2DJ52.1 nucleotide sequence (after codon optimization)

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

以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。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)

1.一种高催化活性L-2-HG脱氢酶,其特征在于,所述高催化活性L-2-HG脱氢酶来自包括但不限于固氮弧菌(Azoarcus olearius)BH72、潘多拉菌(Pandoraea sputorum)NCTC13161和印度杆菌(Indibacter alkaliphilus)LW1中的任意一种或多种;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; 具体的,所述L-2-HG脱氢酶选自:Specifically, the L-2-HG dehydrogenase is selected from: (a1)SEQ ID NO.1-3所示氨基酸序列组成的蛋白质;(a1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1-3; (a2)将(a1)所示的氨基酸序列经过一个或多个氨基酸残基的取代、缺失和/或添加且具有相同或相近功能的蛋白质;(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)与(a1)或(a2)所示的氨基酸序列组成具有同一性达到50%或50%以上并且具有与(a1)或(a2)所示的蛋白质具有相同或相近功能的蛋白质。(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). 2.一种多聚核苷酸,其特征在于,所述多聚核苷酸能够编码权利要求1所述高催化活性L-2-HG脱氢酶。2. A polynucleotide, characterized in that the polynucleotide can encode the high catalytic activity L-2-HG dehydrogenase according to claim 1. 3.如权利要求2所述的多聚核苷酸,其特征在于,所述多聚核苷酸具有(b1)-(b4)中任一所述核苷酸序列:3. The polynucleotide of claim 2, wherein the polynucleotide has any one of the nucleotide sequences (b1)-(b4): (b1)如SEQ ID NO.4-6所示的核苷酸序列;(b1) The nucleotide sequence shown in SEQ ID NO.4-6; (b2)如(b1)所示的核苷酸序列经过一个或多个核苷酸的取代、缺失和/或添加形成的序列;(b2) A sequence formed by the substitution, deletion and/or addition of one or more nucleotides to the nucleotide sequence shown in (b1); (b3)与(b1)或(b2)限定的核苷酸序列具有50%或50%以上的同一性,且编码所述融合蛋白的核酸分子;(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)在严格条件下能够与如(b1)-(b3)中任一项所述核苷酸序列杂交并编码相同功能高催化活性L-2-HG脱氢酶的核苷酸序列;(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; 进一步的,所述多聚核苷酸为DNA,包括cDNA、基因组DNA或重组DNA。Further, the polynucleotide is DNA, including cDNA, genomic DNA or recombinant DNA. 4.一种重组表达载体,其特征在于,所述重组表达载体至少包含权利要求2或3所述多聚核苷酸;4. A recombinant expression vector, characterized in that the recombinant expression vector at least contains the polynucleotide of claim 2 or 3; 进一步的,所述重组表达载体通过权利要求2或3所述多聚核苷酸有效地连接到表达载体上获得,所述表达载体包括病毒载体、质粒、噬菌体、黏粒或人工染色体;进一步的,所述表达载体为质粒,包括pACYCDuet-1质粒。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. 5.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求2或3所述多聚核苷酸、权利要求4所述重组表达载体或者能够表达权利要求1所述高催化活性L-2-HG脱氢酶。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.权利要求1所述高催化活性L-2-HG脱氢酶、权利要求2或3所述多聚核苷酸、权利要求4所述重组表达载体和/或权利要求5所述宿主细胞在制备检测L-2-HG的生物传感器中的应用。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. 7.一种检测L-2-HG的生物传感器,其特征在于,所述生物传感器至少包含权利要求1所述高催化活性L-2-HG脱氢酶,以及氧化还原反应指示剂;其中,所述氧化还原反应指示剂为可接受所述高催化活性L-2-HG脱氢酶催化L-2-HG脱氢产生电子的电子受体;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; 进一步的,所述电子受体包括偶氮间苯四酚、3’-[1-[(苯胺基)-羰基]-3,4-四唑]-双(4-甲氧基-6-硝基)苯-磺酸钠和二氯酚靛酚。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.如权利要求7所述生物传感器,其特征在于,所述生物传感器还包括其他用于L-2-HG检测的试剂、装置和/或设备;8. The biosensor according to claim 7, wherein the biosensor also includes other reagents, devices and/or equipment for L-2-HG detection; 进一步的,所述试剂包括检测缓冲液;Further, the reagent includes a detection buffer; 进一步的,所述检测L-2-HG的生物传感器至少包括:0.1~50μM刃天青、0.005~1mg mL-1AoL2HGDH,以及缓冲液;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; 所述缓冲液选自Tris-HCl、HEPES、磷酸钾、磷酸钠、MOPS以及PBS,所述缓冲液浓度为50~100mM,pH为5.8~9.0;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; 进一步优选的,所述检测L-2-HG的生物传感器包括:67mM PBS(pH 6.6)、1μM刃天青、0.05mg mL-1AoL2HGDH。Further preferably, the biosensor for detecting L-2-HG includes: 67mM PBS (pH 6.6), 1μM resazurin, 0.05mg mL -1 AoL2HGDH. 9.一种检测试剂盒,其特征在于,所述检测试剂盒包含上述检测L-2-HG的生物传感器。9. A detection kit, characterized in that the detection kit contains the above-mentioned biosensor for detecting L-2-HG. 10.一种检测L-2-HG的方法,其特征在于,所述方法至少包括:将待测样品与权利要求7或8所述生物传感器进行孵育,分析L-2-HG的浓度或有无;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; 进一步的,所述待测样品为含有或疑似含有L-2-HG的样品,所述样品为生物样品或环境样品,所述生物样品包括细菌培养基、细菌裂解液、细胞培养基、细胞裂解液、动物血清、动物尿液和动物组织液;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; 更进一步的,所述动物为哺乳动物,所述哺乳动物包括人。Furthermore, the animal is a mammal, and the mammal includes a human.
CN202310936644.0A 2023-07-27 2023-07-27 An L-2-hydroxyglutarate biosensor based on highly catalytically active L-2-hydroxyglutarate dehydrogenase and its application Pending CN116949001A (en)

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