阅读说明：本技术 利用fad依赖型d-2-羟基戊二酸脱氢酶和刃天青检测d-2-羟基戊二酸的方法 (Method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin ) 是由 张文 于 2020-12-07 设计创作，主要内容包括：本发明公开了利用FAD依赖型D-2-羟基戊二酸脱氢酶和刃天青检测D-2-羟基戊二酸的方法,涉及酶法检测技术领域,该方法利用FAD依赖型D-2-羟基戊二酸脱氢酶、刃天青和PBS缓冲液,实现D-2-羟基戊二酸的检测；所述FAD依赖型D-2-羟基戊二酸脱氢酶为AX-F-D2HGDH蛋白或P-D2HGDH蛋白；本发明的检测方法组成成分更加简单,成本更低,操作更为简便；由于无需添加辅因子、辅酶或心肌黄酶,因此引入的干扰少,灵敏度更高,并且仅需制备一种酶,无需添加外源辅因子NAD等,同时缓冲液可采用成本更低且易于获取的PBS缓冲液,因此成本更加低廉。(The invention discloses a method for detecting D-2-hydroxyglutaric acid by using FAD-dependent D-2-hydroxyglutaric dehydrogenase and resazurin, which relates to the technical field of enzymatic detection, and realizes the detection of D-2-hydroxyglutaric acid by using FAD-dependent D-2-hydroxyglutaric dehydrogenase, resazurin and PBS buffer solution; the FAD-dependent D-2-hydroxyglutarate dehydrogenase is AX-F-D2HGDH protein or P-D2HGDH protein; the detection method disclosed by the invention is simpler in component, lower in cost and simpler and more convenient to operate; as the addition of a cofactor, coenzyme or diaphorase is not required, the introduced interference is less, the sensitivity is higher, only one enzyme needs to be prepared, exogenous cofactor NAD and the like do not need to be added, and meanwhile, the buffer solution can adopt PBS buffer solution which is lower in cost and easy to obtain, so the cost is lower.)

1.A method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin, which is characterized by comprising the following steps: detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase, resazurin and PBS buffer solution;

the FAD-dependent D-2-hydroxyglutarate dehydrogenase is AX-F-D2HGDH protein or P-D2HGDH protein;

wherein the encoding gene sequence of the AX-F-D2HGDH protein is SEQ ID NO: 1;

the amino acid sequence of the AX-F-D2HGDH protein is SEQ ID NO: 2;

the coding gene sequence of the P-D2HGDH protein is SEQ ID NO: 3;

the amino acid sequence of the P-D2HGDH protein is SEQ ID NO: 4.

2. the method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 1, characterized in that: the FAD-dependent D-2-hydroxyglutarate dehydrogenase is AX-F-D2HGDH protein.

3. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 1, characterized in that: the method comprises the following steps:

(1) exogenous expression, separation and purification of FAD dependent D-2-hydroxyglutarate dehydrogenase

Selecting coding genes of FAD-dependent D-2-hydroxyglutarate dehydrogenase to synthesize target genes through whole genes by combining sequence comparison analysis means to obtain the FAD-dependent D-2-hydroxyglutarate dehydrogenase target genes;

the FAD-dependent D-2-hydroxyglutarate dehydrogenase target gene obtained in the step is connected into pETDuet-1, transferred into an escherichia coli BL21(DE3) strain, subjected to enzyme digestion verification, and then connected into an LB liquid culture medium, and when OD600 reaches 0.5-0.9, 1mM IPTG is added, and induction is carried out at 22 ℃ and 180rpm for 8-16 hours;

purifying the protein: crushing thalli by adopting ultrasound and high pressure to obtain crude enzyme liquid containing FAD dependent D-2-hydroxyglutarate dehydrogenase target protein; separating and purifying protein by using a rapid protein purification instrument and a nickel affinity column, eluting by using 50% Buffer B, concentrating by using an ultrafiltration tube to obtain the protein with the concentration of 1-20mg/ml, and freezing and storing the obtained protein at-80 ℃;

(2) detection of D-2-hydroxyglutarate Using FAD-dependent D-2-hydroxyglutarate dehydrogenase, Resazurin and PBS buffer

Preparing a working solution:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 0.2-10 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.01-0.03mg/ml to obtain a working solution;

② sample preparation

Serum, urine, cerebrospinal fluid, cell culture medium, interstitial fluid or intracellular fluid are subjected to protein removal treatment to obtain a sample to be detected;

preparing standard product

Preparing a series of D-2-hydroxyglutaric acid standard solutions with the concentration of 0-50 mu M;

when the kit is used for detecting serum, urine or cell culture medium samples, the standard substance needs to be correspondingly dissolved in serum, urine or fresh culture medium of healthy people;

when the kit is used for detecting cerebrospinal fluid, interstitial fluid or intracellular fluid samples, the standard substance is dissolved in the triple distilled water;

when the reagent is used for detecting a deproteinized sample, the prepared standard substance can be used after the same deproteinization operation;

detection

Adding 25 μ L of standard substance and sample to be tested into black 96-well plate, respectively, adding 75 μ L of working solution into each of the three repeat wells, and reacting for 20-30min in dark; measuring the fluorescence intensity by using a fluorescence microplate reader, wherein the excitation wavelength is 540nm, and the emission wavelength is 590 nm;

fifthly, making a standard curve

Taking a standard solution without containing D-2-hydroxyglutaric acid as a control group, and calculating the average fluorescence intensity of the control group; subtracting the average fluorescence intensity of the control group from each standard sample hole to obtain the actual fluorescence intensity, calculating the average value of the concentration of each standard sample, and then taking the concentration of the D-2-HG standard sample as the ordinate and the average fluorescence intensity as the abscissa to make a standard curve;

calculation of D-2-hydroxy glutaric acid content in sample

Taking a standard solution without D-2-hydroxyglutaric acid as a control group, and calculating the average fluorescence intensity of the control group; and subtracting the average fluorescence intensity of the control group from each repeated hole to obtain the actual fluorescence intensity, calculating the average value of the repeated holes, and substituting the average value into the standard curve to obtain the concentration of the D-2-hydroxyglutaric acid of the sample to be detected.

4. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 3, characterized in that: the upstream of the whole gene synthesis target gene is provided with an EcoRI enzyme cutting site, and the downstream is provided with a HindIII enzyme cutting site.

5. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 3, characterized in that:

the preparation working solution comprises the following steps: 0.067M PBS (pH 7.4) is used as a buffer solution, the final concentration of resazurin is added to 10 mu M, FAD-dependent D-2-hydroxyglutarate dehydrogenase is added to the final concentration of 0.03mg/ml, and a working solution is obtained.

6. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 3, characterized in that:

the sample processing operation is as follows:

collecting venous blood, then obtaining serum to be detected by using a serum separation gel procoagulant blood collection tube, and removing protein to obtain a serum sample to be detected;

or centrifuging to obtain supernatant, and removing protein to obtain sample to be tested;

or centrifuging the urine to obtain supernatant, and removing protein to obtain a sample to be detected;

or taking the cell for culture, taking the cell culture medium, centrifuging, collecting the supernatant, and removing protein to obtain a sample to be detected for later use;

or taking supernatant after cell lysis, removing protein to obtain a sample to be detected for later use;

or taking a tissue sample, adding cell lysate, performing ultrasonic homogenization, adding protease K, incubating, determining the protein concentration A by using the kit, and removing protein to obtain a sample to be detected for later use;

or other samples with protein concentration less than 0.2mg/ml, and can be used directly or after being diluted properly.

7. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 3, characterized in that:

and when the concentration of the D-2-HG of the sample to be detected is 0-5 mu M, accurately detecting the concentration of the D-2-HG of the sample to be detected, specifically, adopting working solution as follows:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 1 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.013mg/ml to obtain a working solution; when a standard curve is prepared, the series concentration of the prepared D-2-HG standard solution is between 0 and 5 mu M;

when the concentration of the D-2-HG of the sample to be detected is 0-0.5 mu M, the adopted working solution is as follows:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 0.2 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.01mg/ml to obtain a working solution; when a standard curve is prepared, the series concentration of the prepared D-2-HG standard solution is between 0 and 0.5 mu M.

8. The method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 1, characterized in that:

FAD-dependent D-2-hydroxyglutarate dehydrogenase employs the following coding gene sequence:

firstly, the sequence consistency with the coding gene of FAD-dependent D-2-hydroxyglutarate dehydrogenase is more than 70 percent, and the coding protein has the activity of D-2-hydroxyglutarate dehydrogenase;

secondly, the amino acid sequence of the protein has more than 50 percent of consistency with the FAD-dependent D-2-hydroxyglutarate dehydrogenase, and the encoded protein has the activity of D-2-hydroxyglutarate dehydrogenase.

9. Use of the method of any one of claims 1 to 8 for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin, characterized in that: is used for developing a D-2-hydroxyglutaric acid detection kit.

10. The use of the method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin according to claim 9, characterized in that:

the kit comprises a buffer liquid bottle, a standard sample tube, a resazurin solution, an enzyme solution and an instruction book; wherein the buffer solution bottle is filled with PBS buffer solution for preparing working solution, the standard sample tube is filled with D-2-HG standard sample, the resazurin solution is filled with resazurin prepared by triple distilled water, and the enzyme solution is filled with D-2-hydroxyglutarate dehydrogenase.

Technical Field

The invention relates to the technical field of enzymatic detection, in particular to a method for detecting D-2-hydroxyglutaric acid by using FAD-dependent D-2-hydroxyglutaric dehydrogenase and resazurin.

Background

2-Hydroxyglutarate (2-HG) is generally a reduction product of 2-ketoglutarate, and 2-HG contains an asymmetric carbon atom in the molecule and is classified into D-2-Hydroxyglutarate (D-2-HG or R-2-HG) and L-2-Hydroxyglutarate (L-2-HG or S-2-HG) according to its optical activity, and the enzyme responsible for catalyzing the oxidation of D-2-HG to 2-ketoglutarate in vivo is D-2-Hydroxyglutarate dehydrogenase (D-2-Hydroxyglutarate dehydrogenase; D2 HGDH).

It has been reported in the literature that D2HGDH of 70kg of adult humans can decompose 40 mmol of D-2-HG daily. D2HGDH coding gene mutation can cause the increase of D-2-HG concentration, thus causing D-2-hydroxyglutaranoacidosis (D-2-HGA), the D-2-HGA is an autosomal invisible hereditary disease, patients can suffer from epilepsy, hypotonia, hypoevolutism, nerve function damage and the like in early years, and the prognosis is poor; D-2-HGA is divided into type I and type II, and D2HGDH encoding gene mutation and Isocitrate Dehydrogenase (IDH) encoding gene mutation are molecular mechanisms of type I and type II D-2-HGA occurrence respectively. Human body D2HGDH is covalently bound with FAD as a cofactor, D-2-HG and D-lactic acid can be used as substrates, and electron transfer flavoprotein (ETF for short) is used as a natural electron acceptor. This covalent binding of FAD to the cofactor D2HGDH is referred to herein as FAD-dependent D2HGDH, abbreviated F-D2 HGDH.

The D-2-HG content in the body fluid of the D-2-HGA patient is 30 to 840 times higher than that of a healthy person (about 26 to 757 mu M). D-2-HG is the key to pathogenesis, and the severity of the disease is closely related to the concentration of D-2-HG. Research shows that high concentration D-2-HG has cellular and neurovirulence; accumulation of D-2-HG may down-regulate expression of creatine kinase, complex IV and complex V; inducing oxidative stress, disrupting mitochondrial energy metabolism, and the like. Meanwhile, the concentration of D-2-HG is also increased in various metabolic disturbance diseases such as acyl coenzyme A dehydrogenase deficiency, dihydrolipoic acid dehydrogenase deficiency, pyruvic acid dehydrogenase deficiency and the like. The high concentration of D-2-HG contained in the blood or urine is a biochemical marker of D-2-HGA, so that the development of a sensitive and accurate D-2-HG detection method is of great significance for the diagnosis of the diseases.

In addition, in recent years, Isocitrate Dehydrogenase (IDH) mutations have been detected in a variety of tumors such as acute myeloid leukemia, glioma and glioblastoma multiforme, and IDH mutations cause the protein to have activity of catalyzing the reduction of 2-oxoglutarate to D-2-HG, resulting in the accumulation of D-2-HG. D-2-HG is a competitive inhibitor of numerous 2-oxoglutarate-dependent dioxygenases, including histone demethylases, and its accumulation can lead to genome-wide alteration of histones and DNA, preventing cell differentiation, and ultimately leading to the development of cancer. D-2-HG is also considered to be a carcinogenic organism as a key factor in the development of neoplasia due to IDH mutation. Therefore, the clinical application value of the D-2-HG as the marker draws great attention, for example, the D-2-HG can be used as a substitute marker to predict IDH mutation, meanwhile, compared with a next generation sequencing method, the D-2-HG detection is sensitive and rapid, the IDH mutation false negative caused by a gene sequencing method can be prevented, and the D-2-HG can be used as a glioma typing marker and used as a diagnostic, prognostic and treatment monitoring index and the like in acute myelocytic leukemia and breast cancer.

At present, the concentration of D-2-HG in tissues and body fluids is mainly determined by liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS); the nuclear magnetic method is mainly used for non-invasive detection of D-2-HG in tissues such as glioma and the like. In addition, measuring the total amount of the two metabolites may lead to erroneous conclusions, so that in clinical and scientific research, the determination of D-2-HG and L-2-HG respectively is a prerequisite for accurate diagnosis of 2-HG-related diseases, and is more convincing than the determination of the total amount of the two, and the currently adopted method mainly adopts a chiral separation column or adopts a chiral derivative sample to realize the separation of D-2-HG and L-2-HG. The detection method has the defects of high cost, large labor consumption, complicated steps, difficulty in high flux and the like.

In contrast to F-D2HGDH, in certain anaerobic bacteria, such as Acylaminococcus fermentors, there is an NAD-dependent D-2-hydroxyglutarate dehydrogenase (NAD-dependent D-2-hydroxyglutarate dehydrogenase; named HgdH), which is sometimes also called D2HGDH, in order to avoid confusion with the conventional concept of F-D2HGDH, HgdH is therefore also named N-D2HGDH in the present application. The main function of N-D2HGDH in organisms is to reduce 2-KG to D-2-HG, while oxidizing NADH to NAD. However, in vitro, the N-D2HGDH reaction may also proceed toward the conversion of D-2-HG to 2-ketoglutarate, if facilitated by other enzyme coupling reactions.

The invention patent CN201380011978.1 discloses a tool and a method for measuring (D)2 hydroxyglutaric acid (D2HG) or (D)2 hydroxyadipic acid, and develops a method for detecting D-2-HG by using a combination of NAD dependent dehydrogenase-diaphorase-resazurin, in particular to a method for realizing the final transmission of electrons to the resazurin to generate fluorescence by using the coupling reaction of N-D2HGDH and diaphorase, so as to realize the detection of D-2-HG. However, the method has the advantages that because the method adopts two steps of reactions (the first step is that N-D2HGDH catalyzes D-2 HG for oxidation and reduces NAD to generate NADH, and the second step is that myocardial xanthase oxidizes NADH and reduces Resazurin), the factors influencing the result during detection are more, the two steps of reactions are catalyzed and coupled to realize that final electrons are transmitted to Resazurin to generate fluorescence, the introduced interference is more, and the sensitivity is lower; the detection system needs to use two enzymes and needs to add a cofactor NAD with higher price, so the detection cost is higher; in addition, the NAD-dependent dehydrogenase has activity on D-2-HG and also on D-2-hydroxyadipic acid, the substrate specificity of the enzyme for detection is not good, and the risk of interference in detection is high.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a method for detecting D-2-hydroxyglutarate by using a FAD-dependent D-2-hydroxyglutarate dehydrogenase and a resazurin one-step method and application thereof.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the method for detecting D-2-hydroxyglutaric acid by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin and the detection of D-2-hydroxyglutaric acid by using FAD-dependent D-2-hydroxyglutarate dehydrogenase, resazurin and PBS buffer solution are realized, and the principle is shown in FIG. 1; the FAD-dependent D-2-hydroxyglutarate dehydrogenase is covalently bound with the FAD as a cofactor, and the resazurin is directly used as an electron acceptor without any external electron mediator;

the FAD-dependent D-2-hydroxyglutarate dehydrogenase is AX-F-D2HGDH protein or P-D2HGDH protein;

wherein the encoding gene sequence of the AX-F-D2HGDH protein is SEQ ID NO: 1;

the amino acid sequence of the AX-F-D2HGDH protein is SEQ ID NO: 2;

the coding gene sequence of the P-D2HGDH protein is SEQ ID NO: 3;

the amino acid sequence of the P-D2HGDH protein is SEQ ID NO: 4.

preferably, the FAD-dependent D-2-hydroxyglutarate dehydrogenase is an AX-F-D2HGDH protein.

Preferably, the method for detecting D-2-hydroxyglutarate by using the FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin comprises the following steps:

(1) exogenous expression, separation and purification of FAD dependent D-2-hydroxyglutarate dehydrogenase

Selecting coding genes of FAD-dependent D-2-hydroxyglutarate dehydrogenase to synthesize target genes through whole genes by combining sequence comparison analysis means to obtain the FAD-dependent D-2-hydroxyglutarate dehydrogenase target genes;

the FAD-dependent D-2-hydroxyglutarate dehydrogenase target gene obtained in the step is connected into pETDuet-1, transferred into an escherichia coli BL21(DE3) strain, subjected to enzyme digestion verification, and then connected into an LB liquid culture medium, and when OD600 reaches 0.5-0.9, 1mM IPTG is added, and induction is carried out at 22 ℃ and 180rpm for 8-16 hours;

purifying the protein: crushing thalli by adopting ultrasound and high pressure to obtain crude enzyme liquid containing FAD dependent D-2-hydroxyglutarate dehydrogenase target protein; separating and purifying protein by using a rapid protein purification instrument and a nickel affinity column, eluting by using 50% Buffer B, concentrating by using an ultrafiltration tube to obtain the protein with the concentration of 1-20mg/ml, and freezing and storing the obtained protein at-80 ℃;

(2) detection of D-2-hydroxyglutarate Using FAD-dependent D-2-hydroxyglutarate dehydrogenase, Resazurin and PBS buffer

Preparing a working solution:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 0.2-10 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.01-0.03mg/ml to obtain a working solution;

② sample preparation

Serum, urine, cerebrospinal fluid, cell culture medium, interstitial fluid or intracellular fluid can be subjected to deproteinization treatment to obtain a sample to be detected;

preparing standard product

Preparing a series of D-2-hydroxyglutaric acid standard solutions with the concentration of 0-50 mu M;

when the kit is used for detecting serum, urine or cell culture medium samples, the standard substance needs to be correspondingly dissolved in serum, urine or fresh culture medium of healthy people;

when the kit is used for detecting cerebrospinal fluid, interstitial fluid or intracellular fluid samples, the standard substance is dissolved in the triple distilled water;

when the standard substance is used for detecting deproteinized samples, the prepared standard substances can be used after the same deproteinization operation;

detection

Adding 25 μ L of standard substance and sample to be tested into black 96-well plate, respectively, adding 75 μ L of working solution into each of the three repeat wells, and reacting for 20-30min in dark; measuring the fluorescence intensity by using a fluorescence microplate reader, wherein the excitation wavelength is 540nm, and the emission wavelength is 590 nm;

the sample to be tested and the standard curve are measured together, so that the result is accurate, and the fluctuation of the curve under different conditions caused by the fluctuation of external conditions is prevented;

fifthly, making a standard curve, taking a standard solution without D-2-hydroxyglutaric acid as a control group, and calculating the average fluorescence intensity of the control group; subtracting the average fluorescence intensity of the control group from each standard sample hole to obtain the actual fluorescence intensity, calculating the average value of the concentration of each standard sample, and then taking the concentration of the D-2-HG standard sample as the ordinate and the average fluorescence intensity as the abscissa to make a standard curve;

sixthly, calculating the content of the D-2-hydroxyglutaric acid of the sample, taking a standard solution without the D-2-hydroxyglutaric acid as a reference group, and calculating the average fluorescence intensity of the reference group; and subtracting the average fluorescence intensity of the control group from each repeated hole to obtain the actual fluorescence intensity, calculating the average value of the repeated holes, and substituting the average value into the standard curve to obtain the concentration of the D-2-hydroxyglutaric acid of the sample to be detected.

Preferably, the whole gene synthesis target gene has an EcoRI restriction site at the upstream and a HindIII restriction site at the downstream.

Preferred sample processing operations are:

collecting venous blood, then obtaining serum to be detected by using a serum separation gel procoagulant blood collection tube, and removing protein to obtain a serum sample to be detected;

or centrifuging to obtain supernatant, and removing protein to obtain sample to be tested;

or centrifuging the urine to obtain supernatant, and removing protein to obtain a sample to be detected;

or taking the cell for culture, taking the cell culture medium, centrifuging, collecting the supernatant, and removing protein to obtain a sample to be detected for later use; or taking supernatant after cell lysis, removing protein to obtain a sample to be detected for later use;

or taking a tissue sample, adding cell lysate, performing ultrasonic homogenization, adding protease K, incubating, determining the protein concentration A by using the kit, and removing protein to obtain a sample to be detected for later use;

or other samples with low protein concentration (less than 0.2mg/ml) can be used directly or after proper dilution.

Further, the working solution is: 0.067M PBS (pH 7.4) is used as a buffer solution, the final concentration of resazurin is added to 10 mu M, FAD-dependent D-2-hydroxyglutarate dehydrogenase is added to the final concentration of 0.03mg/ml, and a working solution is obtained.

Further, when the concentration of the D-2-HG of the sample to be detected is 0-5 mu M, the concentration of the D-2-HG of the sample to be detected is accurately detected, and specifically, the working solution is as follows:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 1 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.013mg/ml to obtain a working solution; when a standard curve is prepared, the series concentration of the prepared D-2-HG standard solution is between 0 and 5 mu M.

When the concentration of the D-2-HG of the sample to be detected is 0-0.5 mu M, the working solution is as follows:

adopting 0.067M PBS with pH7.4 as a buffer solution, adding resazurin to a final concentration of 0.2 mu M, and adding FAD-dependent D-2-hydroxyglutarate dehydrogenase to a final concentration of 0.01mg/ml to obtain a working solution; when a standard curve is prepared, the series concentration of the prepared D-2-HG standard solution is between 0 and 0.5 mu M.

Preferably, the FAD-dependent D-2-hydroxyglutarate dehydrogenase employs the following coding gene sequence:

firstly, the sequence consistency with the coding gene of FAD-dependent D-2-hydroxyglutarate dehydrogenase is more than 70 percent, and the coding protein has the activity of D-2-hydroxyglutarate dehydrogenase;

secondly, the amino acid sequence of the protein has more than 50 percent of consistency with the FAD-dependent D-2-hydroxyglutarate dehydrogenase, and the encoded protein has the activity of D-2-hydroxyglutarate dehydrogenase.

The invention also discloses application of the method for detecting D-2-hydroxyglutaric acid by using the FAD-dependent D-2-hydroxyglutaric dehydrogenase and resazurin in development of a D-2-hydroxyglutaric acid detection kit.

Preferably, the kit comprises a buffer solution bottle, a standard sample tube, a resazurin solution, an enzyme solution and an instruction book; wherein the buffer solution bottle is filled with PBS buffer solution for preparing working solution, the standard sample tube is filled with D-2-HG standard sample, the resazurin solution is filled with resazurin prepared by triple distilled water, and the enzyme solution is filled with D-2-hydroxyglutarate dehydrogenase.

Compared with the prior art, the invention has the following advantages:

the method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase (F-D2HGDH) and resazurin takes FAD as a cofactor, can catalyze D-2-HG to be converted into 2-KG, simultaneously transfers electrons to resazurin, does not need an additional electron transfer mediator, and namely the two processes of substrate dehydrogenation and electron transfer to resazurin to generate fluorescence are independently completed by the FAD-dependent D-2-hydroxyglutarate dehydrogenase;

the detection system of the method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin only comprises buffer solution, F-D2HGDH and resazurin, and does not need to add expensive cofactors, coenzyme or diaphorase and the like, and firstly, the components are simpler, the cost is lower, and the operation is simpler and more convenient; secondly, as no cofactors, coenzymes or diaphorase are needed to be added, the introduced interference is less, the sensitivity is higher, the conventional detection limit is 0.14 mu M, and the quantitative limit is 0.47 mu M; thirdly, for low-concentration and ultra-low-concentration D-2-hydroxyglutaric acid samples, accurate detection can be realized by adjusting components in a detection system, the lowest detection limit can reach 0.010 mu M, and the quantification limit can reach 0.034 mu M; fourthly, only one enzyme needs to be prepared, exogenous cofactors NAD and the like do not need to be added, and meanwhile, the buffer solution can adopt PBS buffer solution which is lower in cost and easy to obtain, so that the cost is lower.

Drawings

FIG. 1 is a schematic view showing the detection principle of a method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and Resazurin;

FIG. 2 is a diagram showing the results of enzyme digestion verification of BL21(DE3) -pETDuet-1-AX-F-D2 HGDH;

FIG. 3 is a graph showing the results of AX-F-D2HGDH prepared by SDS-PAGE analysis;

FIG. 4 is a diagram showing the results of selecting fluorescence generated by Resazurin as an electron acceptor versus an electron mediator in the case of catalyzing the dehydrogenation of a substrate by AX-F-D2HGDH protein;

FIG. 5 is a standard curve of the high concentration range D-2-HG (0-50. mu.M);

FIG. 6 is a standard curve for the low concentration range D-2-HG (0-5. mu.M);

FIG. 7 is a standard curve of the ultra-low concentration range D-2-HG (0-0.5. mu.M).

FIG. 8 is a standard curve of D-2-HG (0-50. mu.M) formulated in cell culture medium.

Detailed Description

The invention aims to provide a method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin, which is realized by the following technical scheme:

the FAD-dependent D-2-hydroxyglutarate dehydrogenase AX-F-D2HGDH protein or the P-D2HGDH protein can directly transfer electrons generated by catalyzing dehydrogenation of D-2-HG to Resazurin to generate fluorescence, and the fluorescence can be realized without the existence of any electron transfer agent (including electron transfer flavoprotein ETF, N-methylphenazinyl methyl sulfate and diaphorase). The fluorescent signal is generated by one enzyme of D-2-hydroxyglutarate dehydrogenase rather than by the coupling of two enzyme catalyzed reactions. Because the D-2-hydroxyglutarate dehydrogenase is covalently bound with the FAD, the FAD does not need to be added externally in a detection system, and the detection system only contains the F-D2HGDH and the resazurin, and is simpler.

A new F-D2HGDH (named AX-F-D2HGDH) derived from bacteria (Achromobacter xylosoxidans ATCC 27061; Achromobacter xylosoxidans) is utilized, and compared with F-D2HGDH derived from other sources, the AX-F-D2HGDH has higher substrate specificity, has no catalytic activity on D-malic acid and D-lactic acid and only has activity on D-2-HG, and can meet the requirement of high specificity required by detection, especially for P-D2HGDH derived from Pseudomonas stutzeriA 1501.

A method for detecting D-2-hydroxyglutarate by using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin, comprising the steps of:

preparation of AX-F-D2HGDH protein

1.1 acquisition of Gene sequences

A more common model strain of Achromobacter Achromobacter, Achromobacter xylosoxidans ATCC27061, was named AX-F-D2HGDH from A-F-D2HGDH derived from this Achromobacter.

NCBI database entry Gene ID: 29509945, obtaining the gene sequence (SEQ ID NO: 1, 1413bp) and amino acid sequence (SEQ ID NO: 2, 470aa) of AX-F-D2HGDH in Achromobacter xylosoxidans ATCC 27061.

Using a sequence alignment tool (e.g., Nucleotide Blast in NCBI) well known to researchers in the field, the gene sequences of AX-F-D2HGDH are aligned to obtain a sequence with more than 70% of identity, and the encoded protein has D-2-hydroxyglutarate dehydrogenase activity. Alignment of the amino acid sequence of AX-F-D2HGDH using sequence alignment tools well known to researchers in the art (e.g., Protein Blast in NCBI, etc.) results in a sequence with greater than 50% identity and a Protein with D-2-hydroxyglutarate dehydrogenase activity. These sequences are also suitable for the following experiments.

1.2 Gene expression

Entrusting a biological company to synthesize a target gene through a whole gene, connecting the target gene into an efficient escherichia coli expression vector pETDuet-1 by adopting a conventional molecular cloning means (enzyme digestion, connection, transformation and the like), transferring the efficient escherichia coli pEBL 21(DE3) strain, inoculating the efficient escherichia coli expression vector into an LB liquid culture medium, adopting IPTG (isopropyl-beta-thiogalactoside) for induction expression, adding 1mM IPTG (isopropyl-beta-thiogalactoside) when OD600 reaches 0.5-0.9, and carrying out induction expression at 22 ℃ and 180rpm for 8-12 hours.

1.3 protein purification

Breaking the thalli by a conventional method (ultrasonic and high pressure) to obtain a crude enzyme solution containing the target protein; and (3) separating and purifying the protein by adopting a rapid protein purification instrument and a nickel affinity column according to a conventional scheme. Eluting with 50% Buffer B, concentrating with ultrafiltration tube to obtain protein AX-F-D2HGDH of 1-20mg/ml, and freezing at-80 deg.C.

1.4 preparation and purification of Pseudomonas stutzeri A1501-derived P-D2HGDH Zhang W, Zhang M, Gao C, Zhang Y, Ge Y, Guo S, Guo X, ZHou Z, Liu Q, Zhang Y, Ma C, Tao F, Xu P (2017) Coupling beta-3-phosphoglycerate dehydrogenase and D-2-hydroxyglutamate dehydrogenase driving bacteria I-E7582 synthesis, Proc Natl Acad Sci S114

2. Detection of D-2-hydroxyglutaric acid Using AX-F-D2HGDH protein (P-D2HGDH protein) and Resazurin

2.1 preparing working solution

2.1.1 preparation of buffer, 0.067M PBS buffer solution at pH7.4

2.1.2 preparing 0.1mM to 5mM resazurin with double distilled water as a mother solution, and storing at-80 ℃;

2.1.3 working solutions suitable for the detection of high concentrations of D-2-HG (0-50. mu.M) were freshly prepared: adding AX-F-D2HGDH (P-D2HGDH) into the buffer solution to a final concentration of 0.01-0.05 mg/ml; the final concentration of Resazurin is 5-15. mu.M, 10. mu.M is adopted in the present example; ice bath or 4 ℃;

2.1.4 working solutions suitable for the detection of low concentrations of D-2-HG (0-5. mu.M) were freshly prepared: adding A-F-D2HGDH into a buffer solution to a final concentration of 0.01-0.03 mg/ml; the final concentration of Resazurin is 0.5-1 μ M, and the temperature is controlled in ice bath or 4 ℃;

2.1.5 working solutions suitable for the detection of ultra-low concentrations of D-2-HG (0-0.5. mu.M) were freshly prepared: adding A-F-D2HGDH into a buffer solution to a final concentration of 0.01-0.03 mg/ml; resazurin final concentration 0.1-0.25. mu.M, ice bath or 4 ℃.

2.2 preparing a Standard Curve

D-2-HG standards were prepared.

Preparing a standard substance suitable for detecting high-concentration D-2-HG (0-50 mu M): 0. 0.5, 1, 2.5, 5, 10, 25, 50 μ M.

Preparing a standard substance suitable for detecting low-concentration D-2-HG (0-5 mu M): 0. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 2.5, 5 μ M.

Preparing a standard substance suitable for detecting ultra-low concentration D-2-HG (0-0.5 mu M): 0. 0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.5. mu.M.

2.3 detection step

Adding 10-50 μ L sample into black 96-well plate, adding 75 μ L working solution, and reacting for 10-30min in dark. The fluorescence intensity is measured by a fluorescence microplate reader, the excitation wavelength is 500-560nm, the emission wavelength is 570-610nm, the excitation wavelength is 540nm and the emission wavelength is 590nm are preferred. And then, taking the fluorescence intensity as the ordinate and the TMAO standard substance concentration as the abscissa to draw a standard curve. Other smaller volume well plates (e.g., 384 well plates) may also be scaled down.

3. Preparation of test samples

3.1 serum, urine, cerebrospinal fluid and other biological fluid samples and cell culture medium supernatant samples

3.1.1 routine sampling of serum, urine, cerebrospinal fluid and other biological fluids

3.1.2 centrifuging 13000g of the cell culture medium sample for 5min, and taking the supernatant;

3.1.3 deproteinization

A commercial protein removal kit based on perchloric acid is adopted to remove protein from biological liquid samples such as blood, urine, cerebrospinal fluid and the like and related standard products prepared in the liquid samples according to the kit steps, and the obtained samples can be used for detection.

Or removing proteins larger than 10kDa by using a commercial ultrafiltration tube (10kDa) and centrifuging to obtain a deproteinized sample.

Or adopting an ethanol precipitation method: adding a sample of 0.5ml into 1ml of absolute ethyl alcohol, mixing, centrifuging for 10 minutes at 4000g, taking out a certain volume of supernatant, evaporating by using an oven or a rotary concentrator, and adding a proper amount of triple distilled water for dissolving.

3.1.4 biological liquid sample with low protein content can be diluted by 10-20 times and can be detected without a protein removal step.

3.2 intracellular fluid sample adherent cells: the medium was removed and washed gently with pre-cooled PBS 2 times. Then adding commercial cell lysate, repeatedly freezing and thawing for 3 times, lysing adherent cells, centrifuging at 13000g for 5min to remove cell debris, and measuring the protein concentration of the supernatant by using a BCA protein assay kit. Then the deproteinization procedure was followed as described above.

Suspension of cells: the cell culture was centrifuged at 500g for 5min, the cell pellet was washed 2 times with pre-cooled PBS, then commercial cell lysate was added, freeze-thawing was repeated 3 times, 13000g was centrifuged for 5min to remove cell debris, and the supernatant was assayed for protein concentration with BCA protein assay kit. Then the deproteinization procedure was followed as described above.

3.3 cell culture Medium samples

For adherent cells: cell culture medium is taken, 13000g is centrifuged for 5min, and supernatant is collected for PCA deproteinization operation.

For suspension cells: centrifuging the cell culture at 500g for 5min, collecting the supernatant, centrifuging at 13000g for 5min, collecting the supernatant, and performing PCA deproteinization operation.

3.4 tissue samples

Fresh tissue samples: the tissue samples were mixed according to the ratio of 100mg tissue samples to 500. mu.L of commercial cell lysate, homogenized by sonication in the usual way, 10. mu.L of commercial proteinase K was added and incubated for 2h at 37 ℃. Protein concentration was determined using the BCA protein assay kit. Then the deproteinization procedure was followed as described above.

Paraffin-embedded tissue samples: dewaxing was carried out using the conventional scheme for xylene dewaxing. The deparaffinized tissue was treated as described above for fresh tissue samples.

Adding 25 μ L of sample to be tested (serum, urine, cerebrospinal fluid, intracellular fluid sample, cell culture medium sample, tissue sample) into black 96-well plate, adding 75 μ L of working solution, and reacting in dark for 30 min; measuring the fluorescence intensity by using a fluorescence microplate reader, wherein the excitation wavelength is 540nm, and the emission wavelength is 590 nm; detecting each sample to be detected for 3 times;

and calculating the average fluorescence intensity of the control group, subtracting the average fluorescence intensity of the control group with the standard substance of 0 from each hole to obtain the actual fluorescence intensity, calculating the average value of each hole, and substituting the average value into the standard curve to obtain the D-2-HG concentration of the sample to be detected.

The whole gene synthesis biology company in the invention is the company of the Industrial bioengineering (Shanghai) limited;

nickel affinity columns were purchased from GE Healthcare, HisTrap HP;

desalting columns were purchased from GE Healthcare corporation Hitrap desaling;

phenazine methosulfate available from Sigma-Aldrich under accession number P9625;

diaphorase was purchased from Sigma-Aldrich, cat # D5540;

electron Transfer Flavoprotein (ETF) was obtained according to the papers Zhang W, Zhang M, Gao C, Zhang Y, Ge Y, Guo S, Guo X, Zhou Z, Liu Q, Zhang Y, Ma C, Tao F, Xu P (2017) Coupling beta-3-phosphoglycerate dehydrogenase and d-2-methylglutarate dehydrogenase sources bacterial-serum synthesis.

D-2-HG standards were purchased from Sigma-Aldrich;

the manufacturer and model of the fluorescence microplate reader are Cytation5, BioTek;

the perchloric acid-based deproteinization Kit manufacturer is a Deproteinizing Sample Preparation Kit of Biovision corporation, a product number K808;

the manufacturer of cell lysate is Biyuntian corporation, Cat number P0013;

protease K was manufactured by Biyuntian corporation, cat # ST 533;

SW1353 cells and HT1080 cells were purchased from Wuhan Punuoist Life technologies, Inc.

The invention is further described with reference to specific examples.

Examples

Preparation of AX-F-D2HGDH protein

1.1 acquisition of Gene sequences

A more common model strain of Achromobacter Achromobacter, Achromobacter xylosoxidans ATCC27061, was named AX-F-D2HGDH from A-F-D2HGDH derived from this Achromobacter.

NCBI database entry Gene ID: 29509945, obtaining the gene sequence (SEQ ID NO: 1, 1413bp) and amino acid sequence (SEQ ID NO: 2, 470aa) of AX-F-D2HGDH in Achromobacter xylosoxidans ATCC 27061.

The gene sequence of AX-F-D2HGDH is compared by using a sequence comparison tool (such as Nucleotide Blast in NCBI) well known by researchers in the field, a sequence with the consistency of more than 70 percent is obtained, and the encoded protein has the activity of D-2-hydroxyglutarate dehydrogenase, has higher substrate specificity and only has the activity to D-2-HG. The amino acid sequence of AX-F-D2HGDH is compared by using a sequence comparison tool (such as ProteinBlast in NCBI) well known by researchers in the field, a sequence with the consistency of more than 50 percent is obtained, and the protein has D-2-hydroxyglutarate dehydrogenase activity, has high substrate specificity and only has activity on D-2-HG. These sequences are also suitable for the following experiments.

1.2 Gene expression

(1) Entrusted to Bio Inc. to synthesize the coding gene of AX-F-D2HGDH as a whole gene; during synthesis, the upstream of the gene is provided with an EcoRI enzyme cutting site, and the downstream is provided with a HindIII enzyme cutting site;

(2) the conventional scheme utilizes EcoRI and HindIII to connect genes into pETDuet-1, and the genes are transferred into a strain of escherichia coli BL21(DE 3); the clone was named BL21(DE3) -pETDuet-1-AX-F-D2HGDH as shown in FIG. 2.

(3) BL21(DE3) -pETDuet-1-AX-F-D2HGDH strain is inoculated into LB liquid medium, 1mM IPTG is added when OD600 reaches 0.5-0.9, and induction is carried out at 22 ℃ and 180rpm for 12-16 hours;

(4) centrifuging at 8000g for 10min to collect thallus, suspending each 1000mL thallus with 100mL buffer solution A (pH 7.4, 20mM sodium phosphate, 20mM imidazole, 500mM sodium chloride), crushing thallus with high pressure crusher, centrifuging at 8000g for 40min, and filtering the supernatant with 0.22 μm filter membrane to obtain crude enzyme solution containing target protein;

(5) purification was performed using a rapid protein purifier and a nickel affinity column (5 ml). After the conventional equilibration and loading process, after washing with 10% buffer B (pH 7.4, 20mM sodium phosphate, 500mM imidazole, 500mM sodium chloride), 20% buffer B, and 30% buffer B, 50% of the fractions eluted with buffer B, which contained the protein of interest, were collected, as shown in FIG. 3.

(6) The fractions were concentrated by ultrafiltration tube, and the concentrated protein was passed through desalting column (5ml) to replace desalting buffer (50mM Tris-HCl, pH7.4), and then concentrated to 13mg/ml, and then rapidly frozen in liquid nitrogen, and stored at-80 deg.C.

Furthermore, P-D2HGDH Zhang W, Zhang M, Gao C, Zhang Y, Ge Y, Guo S, Guo X, ZHou Z, Liu Q, Zhang Y, Ma C, Tao F, Xu P (2017) Coupling between D-3-phosphoglycerate dehydrogenase and D-2-hydroglucarate dehydrogenase drivers bacterial-series synthesis, Proc Natl Acl Sci U S114: E7574-E7582 from Pseudomonas stutzeri A1501 were prepared according to the following paper procedures.

Substrate specificity experiments for AX-F-D2HGDH protein and P-D2HGDH protein

The enzyme activities of AX-D2HGDH and P-D2HGDH were measured in 800. mu.L reaction system, each of which contained 50mM Tris-HCl buffer (pH 7.4), 200. mu. M N-methylphenazinium methylsulfate, 100. mu.M 2, 6-dichlorophenoindol, 1mM of the following substrate, and 0.025mg/ml of D2HGDH protein and P-D2HGDH protein. The reaction temperature was 30 ℃ and the change in absorbance at a wavelength of 600nm was measured. As shown in Table 1, AX-F-D2HGDH and P-D2HGDH had high substrate specificity for D-2-HG, except that the D2HGDH protein was inactive to D-malic acid, and the P-D2HGDH protein was active to D-malic acid.

TABLE 1 results of substrate specificity experiments for AX-F-D2HGDH and P-D2HGDH

As can be seen from the results in Table 1, the AX-F-D2HGDH protein was active only on D-2-hydroxyglutarate, and was inactive on D-malate and had higher substrate specificity than P-D2 HGDH.

Selection of Electron mediators in the fluorescence production with Resazurin for AX-F-D2HGDH protein and P-D2HGDH protein

For the case that the AX-F-D2HGDH and P-D2HGDH proteins catalyze D-2-HG dehydrogenation, if electron transfer is promoted by adding an electron transfer mediator, the following experiment is carried out:

the electron mediator of choice is N-methylphenazinyl methyl sulfate, diaphorase or Electron Transfer Flavoprotein (ETF);

preparing a working solution:

1/15M PBS (pH 7.4), AX-FD-D2HGDH (or P-D2HGDH protein) at 0.02mg/ml, Resazurin at 10. mu.M, and an electron mediator (8. mu.M of phenazine methosulfate, or 1U/ml diaphorase, or ETF, an electron transfer flavoprotein at 0.1 mg/ml).

Mu.l of triple-distilled water (control) and 25. mu.l of D-2-HG (5. mu.M, 50. mu.M) were added to a black 96-well plate, and 75. mu.L of the working solution was added thereto and reacted for 20min in the dark. Fluorescence intensity was measured using a fluorescence microplate reader (staining 5, BioTek), excitation wavelength 540nm, emission wavelength 590 nm.

The results for AX-F-D2HGDH protein are shown in FIG. 4, where N-methylphenazinyl methylsulfate severely inhibited the generation of the Resazurin fluorescent signal; the diaphorase and the Electron Transfer Flavoprotein (ETF) have no great influence on the generation of the resazurin fluorescence signal. Similar results also occur with P-D2 HGDH: the generation of a 70% fluorescence signal is inhibited by phenazine methosulfate; the addition of diaphorase and Electron Transfer Flavoprotein (ETF) did not significantly increase the generation of fluorescence signal. The spontaneity of the reaction between the AX-F-D2HGDH (or P-D2HGDH) and the resazurin is more highlighted, and the fact that the AX-F-D2HGDH (or P-D2HGDH) can directly generate fluorescence by taking the resazurin as an electron acceptor when catalyzing substrate dehydrogenation does not need an electron mediator is proved.

2. Detection of D-2-hydroxyglutaric acid Using AX-F-D2HGDH protein and Resazurin

2.1 detection System for D-2-HG in conventional concentration range (0-50 μ M) and Standard Curve

2.1.1 preparation of standards

Preparing a D-2-HG standard substance by using triple distilled water: 0. 0.5, 1, 2.5, 5, 10, 25, 50 μ M.

2.1.2 preparation of working fluid

0.067M PBS (pH 7.4) is used as a buffer solution, resazurin is added to a final concentration of 10 mu M, and AX-F-D2HGDH is added to a final concentration of 0.03mg/ml respectively to obtain working solutions.

2.1.3 detection

Adding 25 μ L of triple-distilled water (as control) and the above D-2-HG standard into black 96-well plate, repeating for 3 times, adding 75 μ L of working solution, and reacting for 20min in dark. Fluorescence intensity was measured using a fluorescent microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.1.4 Standard Curve plotting

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group from each well, and the average value of each concentration was obtained. The concentration of D-2-HG was plotted on the ordinate and the average fluorescence intensity was plotted on the abscissa, and the standard curve y was 0.0006x-0.7433 (R)²0.9903) as shown in fig. 5. This section shows the composition of the detection system and the method for preparing the standard curve for this concentration range only as an example, and the standard curve should be prepared at the same time each time the sample is measured. The standard curve generated by the method is suitable for samples without protein removal.

2.2 detection System for D-2-HG at Low concentration (0-5 μ M) and Standard Curve

2.2.1 preparation of standards

Preparing a D-2-HG standard substance by using triple distilled water: 0. 0.1, 0.2, 0.4, 0.75, 1, 2.5, 5 μ M.

2.2.2 preparation of working solution

Using 0.067M PBS at pH7.4 as a buffer, resazurin was added to a final concentration of 1. mu.M, and AX-F-D2HGDH was added to final concentrations of 0.013mg/ml, respectively, to obtain working solutions.

2.2.3 detection

Add 25. mu.l of triple distilled water (control) and the above D-2-HG standard to a black 96-well plate and repeat 3 times. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 20min with exclusion of light. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.2.4 Standard Curve plotting

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group from each well, and the average value of each concentration was obtained. D-2-HG concentration (. mu.M) was plotted on the ordinate (y) and the average fluorescence intensity was plotted on the abscissaLabel (x) yielding the standard curve y ═ 0.0006x (R)²0.9974), as shown in fig. 6. This section shows the composition of the detection system and the method for preparing the standard curve for this concentration range only as an example, and the standard curve should be prepared at the same time each time the sample is measured. The standard curve generated by the method is suitable for samples without protein removal.

Detection system and standard curve of D-2-HG in ultra-low concentration (0-0.5 mu M) range of 2.3

2.3.1 preparation of standards

Preparing a D-2-HG standard substance by using triple distilled water: 0. 0.025, 0.05, 0.1, 0.4, 0.5. mu.M.

2.3.2 preparing working solution

0.067M PBS (pH 7.4) is used as a buffer solution, resazurin is added to a final concentration of 0.2. mu.M, and AX-F-D2HGDH is added to a final concentration of 0.01mg/ml respectively to obtain a working solution.

2.3.3 detection

Add 25. mu.l of triple distilled water (control) and the D-2-HG standard above to a black 96-well plate and repeat 3. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 20min with exclusion of light. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.3.4 Standard Curve plotting

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group from each well, and the average value of each concentration was obtained. The standard curve y was 0.0008x (R) with D-2-HG concentration (μ M) as ordinate (y) and average fluorescence intensity as abscissa (x)²0.9963) as shown in fig. 7. This section shows the composition of the detection system and the method for preparing the standard curve for this concentration range only as an example, and the standard curve should be prepared at the same time each time the sample is measured. The standard curve generated by the method is suitable for samples without protein removal.

As can be seen from the curve, the linear relation between the concentration of D-2-HG and the fluorescence intensity of the AX-F-D2HGDH protein detection system is good, the method can be used for detecting D-2-HG, and the same result is obtained by the same P-D2HGDH protein;

if the concentration of the D-2-HG in the sample is larger than 50 mu M in the detection process, the sample to be detected can be diluted by the buffer solution and then detected again, and then the content of the D-2-HG in the sample to be detected is calculated according to the dilution times; if the detection system needs to be reconfigured and calculated after re-measurement within 0-5 mu M (particularly within 0-0.5), the detection data can be more accurate, and if the data accuracy is not high, the detection result of the standard curve with 0-50 mu M can be directly used.

In this section, the standard D-2-HG dissolved in triple distilled water is used as an example to show the standard curve preparation method in the conventional concentration, low concentration and ultra-low concentration ranges. The D-2-HG standard dissolved in other mediums (serum, urine and the like of healthy people) can also be used for preparing a standard curve in a conventional concentration range, a low concentration range and an ultra-low concentration range, but the standard needs to be subjected to the same processing operation as the sample. Most applications require only a standard curve for a conventional concentration range.

2.4 detection of the content of D-2-HG in serum Using AX-F-D2HGDH protein

2.4.1 serum Collection

Venous blood is collected by a conventional method, and serum is obtained by using a common serum separation gel procoagulant blood collection tube.

2.4.2 preparation of standards

Preparing a standard product of D-2-HG from the serum of a healthy person A: 0.1, 2.5, 5, 10, 25, 50 μ M.

(serum, etc. requires protein removal procedures, one of which may cause dilution of the sample and the other of which may cause inhibition of the enzymatic reaction).

2.4.3 preparation of samples to be tested

D-2-HG was added to the serum of healthy human B to a final concentration of 0. mu.M, 2.5. mu.M, 10. mu.M, 50. mu.M, and named S1, S2, S3, S4, respectively.

2.4.4 deproteinization

Deproteinization operation was performed according to the kit procedure using a deproteinization kit based on perchloric acid. The sample to be detected and the prepared standard substance are subjected to the same deproteinization operation.

2.4.5 preparation of working solution

0.067M PBS (pH 7.4) is used as a buffer solution, resazurin is added to a final concentration of 10 mu M, and AX-F-D2HGDH is added to a final concentration of 0.03mg/ml respectively to obtain working solutions.

2.4.6 detection

Mu.l of the deproteinized standard and the sample to be tested (replicate 2 times) were added to a black 96-well plate. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 30min under dark conditions. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.4.7 calculating concentration

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group (i.e., serum with 0 as a standard) from each well, and the average value of each well was obtained. The concentration of D-2-HG was plotted on the ordinate (y) and the average fluorescence intensity was plotted on the abscissa (x), giving a standard curve y of 0.0007x (R)²0.9984). And substituting the fluorescence intensity of the sample into the standard curve to obtain the concentration of the sample. The concentration of the S1 sample was measured to be 0. mu.M, the concentration of the S2 sample was measured to be 2.34. mu.M, the concentration of the S3 sample was measured to be 10.06. mu.M, and the concentration of the S4 sample was measured to be 50.25. mu.M.

2.4.8 more accurately determine the concentration of sample S2

The concentration of S2 in the sample was between 0-5. mu.M, so in order to determine the concentration of S2 more accurately, the assay and standards were prepared according to 2.2 and tested again. The method specifically comprises the following steps:

2.4.8.1 Standard preparation

Preparing a standard product of D-2-HG by using the serum of the healthy person A: 0. 0.1, 0.2, 0.4, 0.75, 1, 2.5, 5 μ M.

2.4.8.2 deproteinization

The standard was deproteinized as per 2.4.4.

2.4.8.3 preparing working liquid

As with 2.2.2, the final concentration of resazurin was added to 1. mu.M and AX-F-D2HGDH was added to 0.013mg/ml, respectively, using 0.067M PBS (pH 7.4) as a buffer, to obtain working solutions.

2.4.8.4 detection

Mu.l of the deproteinized standard and the sample to be tested (2 replicates) were added to a black 96-well plate. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 30min under dark conditions. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.4.8.5 calculated concentration

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group (i.e., serum with 0 as a standard) from each well, and the average value of each well was obtained. The concentration of D-2-HG was plotted on the ordinate (y) and the average fluorescence intensity was plotted on the abscissa (x), giving a standard curve y of 0.0007x (R)²0.9903). The concentration of the sample S2 is 2.52 mu M by substituting the fluorescence intensity of the sample into the standard curve, which is closer to the actual value (2.5 mu M), and the result is more accurate.

2.5 detection of D-2-HG content in urine Using P-D2HGDH protein

2.5.1 urine Collection

Collecting urine by a conventional method, and carrying out conventional pretreatment;

2.5.2 preparation of standards

Preparing a standard product of D-2-HG from urine of a healthy person A: 0.5, 10, 25, 50, 100. mu.M. (serum, etc. need protein removal operation, one of them may cause dilution of the sample during protein removal operation, and the other may cause some factors inhibiting enzyme reaction during protein removal operation

2.5.3 preparation of samples to be tested

D-2-HG was added to the urine of healthy person B to a final concentration of 0. mu.M, 20. mu.M, 60. mu.M, and named U1, U2, U3, respectively.

2.5.4 deproteinization

Deproteinization was performed according to the kit procedures using a commercial perchloric acid-based deproteinization kit. The sample to be detected and the prepared standard substance are subjected to the same deproteinization operation.

2.5.5 preparation of working solution

0.067M PBS (pH 7.4) is used as a buffer solution, resazurin is added to a final concentration of 10 mu M, and P-D2HGDH is added to a final concentration of 0.04mg/ml respectively to obtain a working solution.

2.5.6 detection

Mu.l of the deproteinized standard and the sample to be tested (replicate 2 times) were added to a black 96-well plate. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 30min under dark conditions. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.5.7 calculating concentration

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group (i.e., urine with 0 as the standard) from each well, and the average value of each well was obtained. The concentration of D-2-HG was plotted on the ordinate (y) and the average fluorescence intensity was plotted on the abscissa (x), giving a standard curve y of 0.0015x (R)²0.983). And substituting the fluorescence intensity of the sample into the standard curve to obtain the concentration of the sample. The U1 sample concentration was measured to be 0. mu.M, the U2 sample concentration was measured to be 20.92. mu.M, and the U3 sample concentration was measured to be 61.14. mu.M.

2.6 detection of D-2-HG content in fresh cryopreserved tissues

2.6.1 sample treatment

Tissue samples from 6 glioma patients (carrying the IDH1R132H mutation) were taken according to ethical norms. A glioma tissue sample of 100mg was taken, mixed with 500. mu.L of a commercial cell lysate, homogenized ultrasonically by a conventional method, 10. mu.L of a commercial proteinase K (20mg/ml) was added, and incubated at 37 ℃ for 2 hours.

Protein concentration A (mg protein/ml) was determined using the BCA protein assay kit.

And (3) adopting a commercial protein removal kit based on perchloric acid, and performing protein removal operation according to kit steps to obtain a protein-removed sample.

2.6.2 preparation of working fluid and Standard

Working solution and standard were prepared according to the method 2.1 above.

2.6.3 detection

Mu.l of deproteinized standard and test sample (duplicate 2) were added to a black 96-well plate. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 30min under dark conditions. Fluorescence intensity was measured using a fluorescence microplate reader (staining 5, BioTek), excitation wavelength 540nm, emission wavelength 590 nm.

2.6.4 calculate concentration

A calibration curve was prepared according to 2.1 to yield y ═ 0.0006x-0.7349 (R)²＝0.993)。

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group (i.e., triple distilled water with 0 as the standard) from each well, and the average value of each well was obtained. The concentration B (. mu.M) of the treated sample was obtained by substituting the fluorescence intensity of the sample into the standard curve. The tissue content of D-2-HG (pmol/mg protein) was obtained by normalization (dividing B by A), and the mean tissue content of D-2-HG in 6 glioma samples was found to be 132pmol/mg protein.

2.7 detection of D-2-HG content in cell culture Medium

2.7.1 cell culture

SW1353 cells carry the IDH2R172S mutation. HT1080 cells carry the IDH1R132C mutation. Both cells were reported to produce D-2-HG and accumulate in the medium. The cultures were performed in 6-well plates, 3 wells each, according to the manufacturer's protocol.

2.7.2 sample Collection

Both SW1353 cells and HT1080 cells were adherent cells. After culturing for 48h, the cell culture medium was taken, and 13000g was centrifuged for 5min, and the supernatant was collected.

2.7.3 deproteinization operation

And (3) adopting a commercial deproteinizing kit based on perchloric acid to perform deproteinizing operation on the sample to be detected and the standard substance according to kit steps.

2.7.4 Standard preparation

0, 1, 2.5, 5, 10, 25, 50. mu.M standard solutions were prepared in 10% FBS-containing MEM medium (all commercially available from Thermo Co.).

2.7.5 preparing working liquid

0.067M PBS (pH 7.4) is used as a buffer solution, resazurin is added to a final concentration of 10 mu M, and AX-F-D2HGDH is added to a final concentration of 0.03mg/ml respectively to obtain working solutions.

2.7.6 detection

Mu.l of the deproteinized standard and the sample to be tested (replicate 2 times) were added to a black 96-well plate. Then, 75. mu.L of the working solution was added thereto, and the mixture was reacted for 30min under dark conditions. Fluorescence intensity was measured using a fluorescence microplate reader, excitation wavelength 540nm, emission wavelength 590 nm.

2.7.7 calculating concentration

The average fluorescence intensity of the control group was calculated, and the actual fluorescence intensity was obtained by subtracting the average fluorescence intensity of the control group (i.e., the medium with the standard of 0) from each well, and the average value of each well was obtained. The standard curve y was 0.0008x (R2 was 0.999) with the D-2-HG concentration as ordinate (y) and the mean fluorescence intensity as abscissa (x), as shown in fig. 8.

The average fluorescence intensity of the control group (i.e., the serum with the standard of 0) was subtracted from each well to obtain the actual fluorescence intensity, and the average value of each well was determined. And substituting the fluorescence intensity of the sample into the standard curve to obtain the concentration of the sample.

The mean concentration of D-2-HG in SW1353 cell culture medium was determined to be 22.3. mu.M; the average concentration of D-2-HG in HT1080 cell culture medium was 92.9. mu.M.

The average concentration of D-2-HG in the HT1080 cell culture medium is 92.9 mu M, the numerical value is not 0-50 mu M, so that a sample to be detected of the HT1080 cell culture medium is diluted by 2 times, the detection is carried out again, the average fluorescence intensity of a control group is calculated, the average fluorescence intensity of the control group (namely, the serum with the standard substance of 0) is subtracted from each hole to obtain the actual fluorescence intensity, and the average value of each hole is obtained. The average concentration of D-2-HG in HT1080 cell culture medium was 98.3. mu.M, which was determined by substituting the fluorescence intensity of the sample into the standard curve and multiplying by 2.

Sequence listing

<110> secondary Hospital of Shandong university

<120> method for detecting D-2-hydroxyglutarate using FAD-dependent D-2-hydroxyglutarate dehydrogenase and resazurin

<141> 2020-12-07

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1413

<212> DNA

<213> Achromobacter xylosoxidans ATCC27061(Achromobacter xylosoxidans ATCC27061)

<400> 1

atgaccacaa ccgacatcgc ccagcgcctg gtccaggctt tgggccccga caccgtcttc 60

accgccagcg acgacatcgc gccctggctg tccgactggc gcggcttgta caacggccac 120

gcccaggcgg tggtccgccc gcgcaccacc gccgaggtcg ccacctgcct ggcgctctgc 180

aacgaggccg gcgtgccggt ggtgccgcgc ggcggcaaca ccggcctgtg tggcggcgcc 240

acgcccgatg ccgcgcccat caacgtggtc ctcagcctgg accgcatgaa cgccgtgcgc 300

gccatcgaca ccgtcgccaa cacgatggtc gccgaagccg gctgcatcct cggcaacctg 360

cgccgcgcgg cgcaggacgc cggccggctg ctgcccttga gcctggccgc cgaggactcc 420

agccagatcg gcggcaacgt cgccaccaat gccggcggcg tcaacgtggt gcgctacggc 480

atggcgcgcg aactggtgct gggcctggaa gcggtgctgc ccaacggcga gatcttcaac 540

ggcctgcgca ccctgcgcaa ggacaacacc ggctacgacc tcaagcagct gctgatcggc 600

tccgagggca cgctcggcgt catcaccgcc gtggcgctgc gcctgttccc gcgcaccgac 660

gtgcgttccg tggtgctggc cgctgtcgaa tccccggccc aggccctgca attgttcgaa 720

atcctgttcg aacaatgcgg cgcccgcttg caggccttcg agtatttttc cggcgactgc 780

ctcgacctgg tcctggccca tgccgcgggc gtgcaagagc cgttcggcca acgttatccg 840

gcctacgtgc tggtcgaact ggccgacacg gccgacgaag ccgccctgac cgcgctgctg 900

gagaacgtga tcggcaccgc gctcgaccgc ggcctgtgcc tggatgccgc cgtctcggcc 960

tcgctggccc agttgcaggc gctgtggaaa ctgcgcgagg aaatctccga agcgcagcgc 1020

gccgacggtc cgcatctcaa gcatgacgtg tcgctgccga tcgaacgcat ccccgacttc 1080

atggtttccg ccgaagcgcg cgtacgcgcg ctgtacccgg acatccgccc cttcatcttc 1140

ggccacttcg gcgacggcaa cctgcattac aacctctcgc gtcccgccgg cgccgaccgc 1200

ggctgggtgg ccgaacacgg tgccgcgatc accgacgcgg tgctcgacga ggtcaaccgg 1260

tacgggggca gcatcagcgc ggaacacggc atcgggcagc tcaagcgcga ccacttcctg 1320

catagcaagg atgcggtgga gttgcggttg atgcgggaga tcaagcgggt gctggatccc 1380

aaggggatca tgaatcccgg aaagctgctg tag 1413

<210> 2

<211> 470

<212> PRT

<213> Achromobacter xylosoxidans ATCC27061(Achromobacter xylosoxidans ATCC27061)

<400> 2

Met Thr Thr Thr Asp Ile Ala Gln Arg Leu Val Gln Ala Leu Gly Pro

1 5 10 15

Asp Thr Val Phe Thr Ala Ser Asp Asp Ile Ala Pro Trp Leu Ser Asp

20 25 30

Trp Arg Gly Leu Tyr Asn Gly His Ala Gln Ala Val Val Arg Pro Arg

35 40 45

Thr Thr Ala Glu Val Ala Thr Cys Leu Ala Leu Cys Asn Glu Ala Gly

50 55 60

Val Pro Val Val Pro Arg Gly Gly Asn Thr Gly Leu Cys Gly Gly Ala

65 70 75 80

Thr Pro Asp Ala Ala Pro Ile Asn Val Val Leu Ser Leu Asp Arg Met

85 90 95

Asn Ala Val Arg Ala Ile Asp Thr Val Ala Asn Thr Met Val Ala Glu

100 105 110

Ala Gly Cys Ile Leu Gly Asn Leu Arg Arg Ala Ala Gln Asp Ala Gly

115 120 125

Arg Leu Leu Pro Leu Ser Leu Ala Ala Glu Asp Ser Ser Gln Ile Gly

130 135 140

Gly Asn Val Ala Thr Asn Ala Gly Gly Val Asn Val Val Arg Tyr Gly

145 150 155 160

Met Ala Arg Glu Leu Val Leu Gly Leu Glu Ala Val Leu Pro Asn Gly

165 170 175

Glu Ile Phe Asn Gly Leu Arg Thr Leu Arg Lys Asp Asn Thr Gly Tyr

180 185 190

Asp Leu Lys Gln Leu Leu Ile Gly Ser Glu Gly Thr Leu Gly Val Ile

195 200 205

Thr Ala Val Ala Leu Arg Leu Phe Pro Arg Thr Asp Val Arg Ser Val

210 215 220

Val Leu Ala Ala Val Glu Ser Pro Ala Gln Ala Leu Gln Leu Phe Glu

225 230 235 240

Ile Leu Phe Glu Gln Cys Gly Ala Arg Leu Gln Ala Phe Glu Tyr Phe

245 250 255

Ser Gly Asp Cys Leu Asp Leu Val Leu Ala His Ala Ala Gly Val Gln

260 265 270

Glu Pro Phe Gly Gln Arg Tyr Pro Ala Tyr Val Leu Val Glu Leu Ala

275 280 285

Asp Thr Ala Asp Glu Ala Ala Leu Thr Ala Leu Leu Glu Asn Val Ile

290 295 300

Gly Thr Ala Leu Asp Arg Gly Leu Cys Leu Asp Ala Ala Val Ser Ala

305 310 315 320

Ser Leu Ala Gln Leu Gln Ala Leu Trp Lys Leu Arg Glu Glu Ile Ser

325 330 335

Glu Ala Gln Arg Ala Asp Gly Pro His Leu Lys His Asp Val Ser Leu

340 345 350

Pro Ile Glu Arg Ile Pro Asp Phe Met Val Ser Ala Glu Ala Arg Val

355 360 365

Arg Ala Leu Tyr Pro Asp Ile Arg Pro Phe Ile Phe Gly His Phe Gly

370 375 380

Asp Gly Asn Leu His Tyr Asn Leu Ser Arg Pro Ala Gly Ala Asp Arg

385 390 395 400

Gly Trp Val Ala Glu His Gly Ala Ala Ile Thr Asp Ala Val Leu Asp

405 410 415

Glu Val Asn Arg Tyr Gly Gly Ser Ile Ser Ala Glu His Gly Ile Gly

420 425 430

Gln Leu Lys Arg Asp His Phe Leu His Ser Lys Asp Ala Val Glu Leu

435 440 445

Arg Leu Met Arg Glu Ile Lys Arg Val Leu Asp Pro Lys Gly Ile Met

450 455 460

Asn Pro Gly Lys Leu Leu

465 470

<210> 3

<211> 1395

<212> DNA

<213> Pseudomonas stutzeri A1501(Pseudomonas stutzeri A1501)

<400> 3

atgaccgacc ccgccctgat cgatgagctg aaaaccctgg tcgagcccgg caaagtgctg 60

accgacgccg attcgctgaa cgcctacggc aaggactgga ccaagcattt cgctcccgcg 120

ccgtcggcca tcgtgttccc caagagcatc gagcaggtgc aggccatcgt gcgctgggcc 180

aacgcccaca aggtcgcgct ggtgccgtcg ggcggtcgca ccggactctc ggccgccgcc 240

gtggcagcca atggcgaggt ggtggtgtct ttcgactaca tgaaccagat tctcgaattc 300

aacgagatgg atcgcaccgc cgtctgccag cccggcgtgg tcaccgcgca gctgcagcag 360

ttcgccgagg acaagggcct gtactacccg gtggacttcg cctccgccgg ctccagtcag 420

atcggcggca acatcggcac caatgccggt ggcatcaagg tgatccgcta cggcatgacc 480

cgcaactggg tcgccggcat gaaggtggta accggcaagg gcgacctgct ggagctgaac 540

aaggacctga tcaagaacgc caccggctac gacctgcgcc agctgttcat cggcgccgag 600

ggcacgctgg gcttcgttgt cgaggccacc atgcgcctgg agcgtcagcc gaccaacctc 660

accgcgctgg tgctgggcac ccccgatttc gattcgatca tgccggtgct gcacgcgttc 720

caggacaagc tggacctgac cgccttcgaa ttcttctccg acaaggcgct ggccaaggtg 780

ctcggccgcg gtgacgtgcc ggcgccgttc gagaccgact gcccgttcta cgcgctgctg 840

gaattcgagg ccaccaccga ggagcgtgcc gagcaggcgc tggcgacctt cgaacattgc 900

gtcgagcagg gctgggtgct cgacggcgtg atgagccaga gcgagcagca gctgcagaac 960

ctgtggaagc tgcgcgagta catctccgag accatcagtc actggacacc gtacaagaac 1020

gacatctcgg tcaccgtcgg caaggtgccg gccttcctca aggaaatcga tgcgatcgtc 1080

ggcgaacact acccggactt cgagatcgtc tggttcggcc atatcggcga cggcaacctg 1140

cacctgaaca tcctcaagcc cgacgccatg gacaaggacg agttcttcgg caagtgcgcg 1200

acggtgaaca aatgggtgtt cgagaccgtg cagaagtaca acggctcgat ctccgccgaa 1260

cacggcgtcg gcatgaccaa acgtgactac ctcgagtaca gccgctcgcc ggcggaaatc 1320

gaatacatga aagcggtcaa ggcggtgttc gatccgaacg gcatcatgaa ccccggcaag 1380

atcttcgcgg cctga 1395

<210> 4

<211> 464

<212> PRT

<213> Pseudomonas stutzeri A1501(Pseudomonas stutzeri A1501)

<400> 4

Met Thr Asp Pro Ala Leu Ile Asp Glu Leu Lys Thr Leu Val Glu Pro

1 5 10 15

Gly Lys Val Leu Thr Asp Ala Asp Ser Leu Asn Ala Tyr Gly Lys Asp

20 25 30

Trp Thr Lys His Phe Ala Pro Ala Pro Ser Ala Ile Val Phe Pro Lys

35 40 45

Ser Ile Glu Gln Val Gln Ala Ile Val Arg Trp Ala Asn Ala His Lys

50 55 60

Val Ala Leu Val Pro Ser Gly Gly Arg Thr Gly Leu Ser Ala Ala Ala

65 70 75 80

Val Ala Ala Asn Gly Glu Val Val Val Ser Phe Asp Tyr Met Asn Gln

85 90 95

Ile Leu Glu Phe Asn Glu Met Asp Arg Thr Ala Val Cys Gln Pro Gly

100 105 110

Val Val Thr Ala Gln Leu Gln Gln Phe Ala Glu Asp Lys Gly Leu Tyr

115 120 125

Tyr Pro Val Asp Phe Ala Ser Ala Gly Ser Ser Gln Ile Gly Gly Asn

130 135 140

Ile Gly Thr Asn Ala Gly Gly Ile Lys Val Ile Arg Tyr Gly Met Thr

145 150 155 160

Arg Asn Trp Val Ala Gly Met Lys Val Val Thr Gly Lys Gly Asp Leu

165 170 175

Leu Glu Leu Asn Lys Asp Leu Ile Lys Asn Ala Thr Gly Tyr Asp Leu

180 185 190

Arg Gln Leu Phe Ile Gly Ala Glu Gly Thr Leu Gly Phe Val Val Glu

195 200 205

Ala Thr Met Arg Leu Glu Arg Gln Pro Thr Asn Leu Thr Ala Leu Val

210 215 220

Leu Gly Thr Pro Asp Phe Asp Ser Ile Met Pro Val Leu His Ala Phe

225 230 235 240

Gln Asp Lys Leu Asp Leu Thr Ala Phe Glu Phe Phe Ser Asp Lys Ala

245 250 255

Leu Ala Lys Val Leu Gly Arg Gly Asp Val Pro Ala Pro Phe Glu Thr

260 265 270

Asp Cys Pro Phe Tyr Ala Leu Leu Glu Phe Glu Ala Thr Thr Glu Glu

275 280 285

Arg Ala Glu Gln Ala Leu Ala Thr Phe Glu His Cys Val Glu Gln Gly

290 295 300

Trp Val Leu Asp Gly Val Met Ser Gln Ser Glu Gln Gln Leu Gln Asn

305 310 315 320

Leu Trp Lys Leu Arg Glu Tyr Ile Ser Glu Thr Ile Ser His Trp Thr

325 330 335

Pro Tyr Lys Asn Asp Ile Ser Val Thr Val Gly Lys Val Pro Ala Phe

340 345 350

Leu Lys Glu Ile Asp Ala Ile Val Gly Glu His Tyr Pro Asp Phe Glu

355 360 365

Ile Val Trp Phe Gly His Ile Gly Asp Gly Asn Leu His Leu Asn Ile

370 375 380

Leu Lys Pro Asp Ala Met Asp Lys Asp Glu Phe Phe Gly Lys Cys Ala

385 390 395 400

Thr Val Asn Lys Trp Val Phe Glu Thr Val Gln Lys Tyr Asn Gly Ser

405 410 415

Ile Ser Ala Glu His Gly Val Gly Met Thr Lys Arg Asp Tyr Leu Glu

420 425 430

Tyr Ser Arg Ser Pro Ala Glu Ile Glu Tyr Met Lys Ala Val Lys Ala

435 440 445

Val Phe Asp Pro Asn Gly Ile Met Asn Pro Gly Lys Ile Phe Ala Ala

450 455 460