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CN110777188A - Method for detecting content of glutamic acid by enzyme method and application thereof - Google Patents

Method for detecting content of glutamic acid by enzyme method and application thereof Download PDF

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CN110777188A
CN110777188A CN201911053004.5A CN201911053004A CN110777188A CN 110777188 A CN110777188 A CN 110777188A CN 201911053004 A CN201911053004 A CN 201911053004A CN 110777188 A CN110777188 A CN 110777188A
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glutamic acid
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enzyme
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花强
朱虹
董辉
金维荣
邓伟伟
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Shanghai Qianghao Biotechnology Co Ltd
Shenyou Genomics Research Institute Nanjing Co Ltd
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Shenyou Genomics Research Institute Nanjing Co Ltd
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Abstract

The invention relates to a method for detecting glutamic acid content by an enzyme method and application thereof, and glycine transaminase AT is utilized 2And a 3-phosphoglycerate dehydrogenase serA double-enzyme coupling system, and determining the content of glutamic acid; the method for detecting the content of the glutamic acid by the enzyme method overcomes the defect that the traditional glutamate dehydrogenase method is not suitable for high-concentration ammonia environment, and creatively uses glycine transaminase AT 2The invention is coupled with 3-phosphoglycerate dehydrogenase serA double enzyme, deaminates the glutamic acid to form α -ketoglutaric acid, and rapidly and correctly characterizes the glutamic acid content in the system by reducing the NADH content through the reduction reaction of α -ketoglutaric acid.

Description

Method for detecting content of glutamic acid by enzyme method and application thereof
Technical Field
The invention relates to the technical field of enzymatic detection, in particular to a method for detecting glutamic acid content by an enzymatic method and application thereof.
Background
The existing methods for detecting glutamic acid mainly comprise liquid chromatography, ion chromatography, spectrophotometry and the like. The liquid chromatography and the ion chromatography have high analysis sensitivity and good separation effect, and are suitable for analysis and measurement of complex samples, but in the method for measuring glutamic acid by using the liquid chromatography, the samples need to be subjected to derivatization treatment, and instruments of the liquid chromatography and the ion chromatography are expensive, time-consuming and high in cost, so that the method is not suitable for analysis and measurement of high-throughput samples.
A detection system for determining the content of glutamic acid by using a traditional spectrophotometry is mainly based on glutamate dehydrogenase, and the method has the advantages of simple operation, low cost and rapid detection. However, when the method is used for detecting blood samples, urine samples and other samples containing high-concentration ammonia, the normal reaction is inhibited due to the presence of the high-concentration ammonia in the system, and accurate results of the content of the glutamic acid in the samples cannot be obtained. This limits the use of glutamate dehydrogenase and its kits in the above samples.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for detecting the content of glutamic acid by an enzyme method and application thereof, so as to solve the problems in the prior art.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for detecting glutamic acid content by enzyme method, using glycine transaminase AT 2And a 3-phosphoglycerate dehydrogenase SerA double-enzyme coupling system to realize the determination of the content of glutamic acid;
wherein glycine transaminase AT 2The nucleotide sequence of (a) is:
ATGTATCAGGAAAGGCTCTTCACACCAGGACCTGTTGAGATCCCCGATAGAGTAAGGGAAGCTCTGGGAAGGCAGATTATCCATCACAGGACAGAAGAGTTCAGGCGCGCCTTTTTGGAAGTGCGAGAACTCCTCAAGAGACTGCTGGATGACCCATCAGAGAACTTCGTCTTTTTCTCATCTTCGGGCACAGGTGCCATGGAAGCGGCTATTCTGAACTTCTTTGAAGAAGGTCAGAAGGTGCTGGTGGTAAACGGCGGAAAGTTTGGCGAGAGATGGTTTTTGCTGGCAAAACACTGGGGGCTTGAGGTTGTGGAGTACAGACTGGACTGGGGCAAGTCCGCAGACCCCGAAAAAGTCAAGGATCTGCTTAAAAAGCATCCCGATTGCAAGGGTGTGCTTCTTCAAATATCAGAAACATCTACAGGTGCTTACCACCCTGTTGAAGATATAGCGGAGGTTTGCAAAAGTGCAGACGCTCTTTTGGTAGCTGATGCCATAACAGCCTTGGGAGTTTATAACTTAAAGCCTTCGGTGGGCATTGATGTAATGGTAGGTGGGTCCCAGAAGGCTCTTATGCTTCCACCTGGACTTTCTCTCCTCTGGTTCTCTCAAAAGGCAAAAGAGAGATTAAAAGACAGAGCCTTTTACTTTAGCGTAAAAAAGGAGCTTGGCAAACAGCAAGAAGGACAGACCGCGTGGACTCCTGCCATAAGCCTCCTTTTAGCTCTGAAGGAGTCTCTTAGTCTTCTTTTGCAAGAGGGAATGGAGAGAGTAGAAAAAAGGTACAGAGCCATGTCGGAGGGAACTAAGAGAGCGATAAGTGCCTTTGGTCTTGAGGTCTTCCCAGAAAGACCAGCCATATCCATAACGGCTGTAAAAAGCGACGATGCGGAAAGGATAAGAAAGGAGCTCTTAAGACATGGTATAAGGATTGCTGGAGGACAGGACCACCTTAAAGGTAAGATTTTCAGGGTTTCCCACATGGGAGTAAGTGAAAAAGATATGCTCATGCTGATAGGTGTGCTGGAGGTAGTACTAAAAAGGCTTGGTTACCCTGTGGAGCTTGGCAGCGGTGTTTGTAGATACTCACAAACTCTAGTAGAATTTGGGCTATGGTAA；
glycine aminotransferase AT 2The amino acid sequence of (a) is: MYQERLFTPGPVEIPDRVREALGRQIIHHRTEEFRRAFLEVRELLKRLLDDPSENFVFFSSSGTGAMEAAILNFFEEGQKVLVVNGGKFGERWFLLAKHWGLEVVEYRLDWGKSADPEKVKDLLKKHPDCKGVLLQISETSTGAYHPVEDIAEVCKSADALLVADAITALGVYNLKPSVGIDVMVGGSQKALMLPPGLSLLWFSQKAKERLKDRAFYFSVKKELGKQQEGQTAWTPAISLLLALKESLSLLLQEGMERVEKRYRAMSEGTKRAISAFGLEVFPERPAISITAVKSDDAERIRKELLRHGIRIAGGQDHLKGKIFRVSHMGVSEKDMLMLIGVLEVVLKRLGYPVELGSGVCRYSQTLVEFGLW, respectively;
the nucleotide sequence of the 3-phosphoglycerate dehydrogenase serA is as follows: ATGGCAAAGGTATCGCTGGAGAAAGACAAGATTAAGTTTCTGCTGGTAGAAGGCGTGCACCAAAAGGCGCTGGAAAGCCTTCGTGCAGCTGGTTACACCAACATCGAATTTCACAAAGGCGCGCTGGATGATGAACAATTAAAAGAATCCATCCGCGATGCCCACTTCATCGGCCTGCGATCCCGTACCCATCTGACTGAAGACGTGATCAACGCCGCAGAAAAACTGGTCGCTATTGGCTGTTTCTGTATCGGAACAAACCAGGTTGATCTGGATGCGGCGGCAAAGCGCGGGATCCCGGTATTTAACGCACCGTTCTCAAATACGCGCTCTGTTGCGGAGCTGGTGATTGGCGAACTGCTGCTGCTATTGCGCGGCGTGCCGGAAGCCAATGCTAAAGCGCACCGTGGCGTGTGGAACAAACTGGCGGCGGGTTCTTTTGAAGCGCGCGGCAAAAAGCTGGGTATCATCGGCTACGGTCATATTGGTACGCAATTGGGCATTCTGGCTGAATCGCTGGGAATGTATGTTTACTTTTATGATATTGAAAATAAACTGCCGCTGGGCAACGCCACTCAGGTACAGCATCTTTCTGACCTGCTGAATATGAGCGATGTGGTGAGTCTGCATGTACCAGAGAATCCGTCCACCAAAAATATGATGGGCGCGAAAGAAATTTCACTAATGAAGCCCGGCTCGCTGCTGATTAATGCTTCGCGCGGTACTGTGGTGGATATTCCGGCGCTGTGTGATGCGCTGGCGAGCAAACATCTGGCGGGGGCGGCAATCGACGTATTCCCGACGGAACCGGCGACCAATAGCGATCCATTTACCTCTCCGCTGTGTGAATTCGACAACGTCCTTCTGACGCCACACATTGGCGGTTCGACTCAGGAAGCGCAGGAGAATATCGGCCTGGAAGTTGCGGGTAAATTGATCAAGTATTCTGACAATGGCTCAACGCTCTCTGCGGTGAACTTCCCGGAAGTCTCGCTGCCACTGCACGGTGGGCGTCGTCTGATGCACATCCACGAAAACCGTCCGGGCGTGCTAACTGCGCTGAACAAAATCTTCGCCGAGCAGGGCGTCAACATCGCCGCGCAATATCTGCAAACTTCCGCCCAGATGGGTTATGTGGTTATTGATATTGAAGCCGACGAAGACGTTGCCGAAAAAGCGCTGCAGGCAATGAAAGCTATTCCGGGTACCATTCGCGCCCGTCTGCTGTACTAA, respectively;
the amino acid sequence of 3-phosphoglycerate dehydrogenase serA is: MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGALDDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGTNQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPEANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAESLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPENPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASKHLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEAQENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHIHENRPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY are provided.
Preferably, the method for detecting the content of the glutamic acid by the enzyme method comprises the following steps:
exogenous expression and separation and purification of protein:
(1) selecting glycine transaminase AT by combining sequence alignment analysis means 2Then respectively synthesizing target gene by using whole gene and PCR to obtain target gene (in which the serA gene clone template is the genome of Escherichia coli E. coli k-12 strain), respectively obtaining glycine transaminase AT 2A target gene and a 3-phosphoglycerate dehydrogenase serA target gene;
(2) subjecting the glycine transaminase AT obtained in step (1) to 2A gene of interest and3-phosphoglycerate dehydrogenase serA target gene is respectively connected into pET28a expression vector by adopting molecular cloning, and pET28a-AT with correct sequencing after cloning 2After being transferred into the Escherichia coli Rosetta (DE3) strain together with pET28a-serA plasmid, the strain was cultured overnight at 37 ℃ in a test tube containing Luria-Bertani (LB) medium, and then transferred into a flask containing LB medium to OD 220 rpm at 37 ℃ in order to obtain a mixture 600AT about 0.6, IPTG was added to a final concentration of 1mM, and the expression was induced AT 37 ℃ for 4 hours to obtain glycine transaminase AT 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
(3) separating and purifying the target protein: centrifuging the bacterial liquid containing the target protein AT low temperature to collect thalli, crushing the thalli, centrifuging to collect supernatant, separating and purifying the target protein by adopting a nickel column for later use, wherein the target protein comprises glycine transaminase AT obtained in the step (2) 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
(II) detecting glutamic acid by a double-enzyme coupling method:
(1) preparing a reaction mixed solution:
preparing 100 mM TEA-HCl solution (pH 9.0), 100 mM sodium glyoxylate solution and 5 mM NADH solution for later use;
configuration of an AT-containing Glycine transaminase 2500. mu.l of a mixture of 3-phosphoglycerate dehydrogenase serA and purified AT AT 3.4 mg/ml in 100. mu.l 2Adding the target protein and 80 mu l of the purified serA target protein with the concentration of 32 mg/ml, and then adding 320 mu l of buffer solution B to make up to 500 mu l for later use;
the composition of buffer B was: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200ml of glycerol, 0.154 g of dithiothreitol, and the pH was adjusted to 7.9 with hydrochloric acid.
Preparing reaction mixed liquor according to the solution, wherein the proportion of each 75 mul of reaction mixed liquor comprises: TEA-HCl solution, 25. mu.l; 10-30 mul of sodium glyoxylate aqueous solution; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; make up to 75. mu.l of ultrapure water.
(2) Preparing a glutamic acid concentration standard curve by an end-point method:
preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mul of glutamic acid standard solutions with each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mul of reaction mixed solution, gently mixing the glutamic acid standard solutions, reacting for 15-30 minutes at 30-40 ℃, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings. Drawing a glutamic acid concentration standard curve by taking the absorbance change value as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate;
(3) initial velocity method for preparing standard curve of glutamic acid concentration
Preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mu l of each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, gently mixing the glutamic acid standard solutions, and reacting for 2-10 minutes at the temperature of 30-40 ℃. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader. And drawing a glutamic acid concentration standard curve by taking the absorbance change value at 340nm within 2-10 minutes as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate.
(III) detection of glutamic acid in urine, blood, or general biological samples:
(1) urine treatment and detection
End-point method: treating urine by using a deproteinization kit, centrifuging to obtain supernatant, and obtaining a urine sample; and during detection, adding 25 mu l of urine into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, slightly and uniformly mixing, reacting for 15-30 minutes at 30-40 ℃, and then finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; urine 25 μ l, make up ultrapure water to 100 μ l. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings.
And calculating the glutamic acid content of the urine according to the change value of the absorbance of the urine sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: treating urine by using a deproteinization kit, centrifuging to obtain supernatant, and obtaining a urine sample; when in detection, 25 mul of urine is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed solution, the mixture is mixed gently and reacts for 2 to 10 minutes at the temperature of 30 to 40 ℃, and the proportion of the reaction mixed solution of a blank control comprises: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the urine according to the change value of the absorbance of the urine sample within 2-10 minutes and a glutamic acid standard curve prepared by an initial velocity method.
(2) Blood treatment and detection
The blood sample used in the present invention is stored in an anticoagulation tube, so that the blood sample contains the anticoagulant of the blood sample, and the anticoagulant in the anticoagulation tube is 10% potassium oxalate-sodium fluoride.
When a blood sample is detected, the blood sample needs to be pretreated, and the steps are as follows: the blood in the anticoagulation tube needs to be firstly centrifuged at low speed to obtain plasma under the condition of 4 ℃ and 4000 rpm for 10 min, and then centrifuged at high speed to remove impurities which can interfere with the experiment from the plasma under the condition of 4 ℃ and 14000 rpm for 10 min. And (4) centrifuging, and taking the supernatant to measure the content of glutamic acid.
End-point method: separating blood to obtain plasma or serum to obtain blood sample; during detection, 25 mul of blood plasma or serum is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed liquid, the mixture is mixed gently and evenly, the reaction is finished after 15-30 minutes of reaction at 30-40 ℃, and the reaction mixed liquid of a blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; plasma or serum 25 μ l, make up ultrapure water to 100 μ l. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings.
And calculating the glutamic acid content of the blood according to the change value of the absorbance of the plasma or serum sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: separating blood to obtain plasma or serum to obtain blood sample; when in detection, 25 mul of blood plasma or serum is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed liquid, the mixture is mixed gently and reacts for 2 to 10 minutes at the temperature of 30 to 40 ℃, and the proportion of the reaction mixed liquid of a blank control comprises: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the plasma or serum sample according to the change value of the absorbance of the plasma or serum sample within 2-10 minutes and a glutamic acid standard curve prepared by an initial velocity method.
(3) Detection of biological samples in general
End-point method: after a general biological sample is treated, taking supernatant, adding 25 mul of biological sample into a 96-hole enzyme label plate filled with 75 mul, mixing the biological sample and the enzyme label plate gently, reacting for 15-30 minutes at 30-40 ℃, and finishing the reaction, wherein the blank control reaction mixed solution comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; typically 25. mu.l of biological sample is supplemented with ultrapure water to 100. mu.l. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings. And calculating the glutamic acid content according to the change value of the absorbance of the general biological sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: after a general biological sample is treated, taking supernatant, adding 25 mul of biological sample into a 96-hole enzyme label plate filled with 75 mul, mixing the biological sample and the enzyme label plate gently, reacting for 2-10 minutes at 30-40 ℃, wherein the reaction mixed solution of a blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the plasma or serum sample according to the change value of the absorbance of the general biological sample within 2-10 minutes and a glutamic acid standard curve prepared by an initial velocity method.
Preferably, the step of separating and purifying the target protein comprises:
A. and (3) centrifuging the bacterial liquid containing the target protein at low temperature, removing supernatant, collecting thalli, adding a buffer solution A, and suspending the thalli in the buffer solution A. The centrifugal condition is that the reaction temperature is 4-16 ℃, and the rotating speed is 10000-12000 rpm; buffer a consisted of the following components: 1L of buffer A contained 2.42 g of tris, 37.3 g of potassium chloride and 100 ml of glycerol, and the pH was adjusted to 7.9 with hydrochloric acid. The target protein comprises glycine transaminase AT obtained in the step (2) 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
B. after the cells are crushed by using a high-pressure cell crusher, centrifuging for 30-50 min at the temperature of 4-16 ℃ and the rpm of 10000-12000, and collecting supernatant;
C. enabling the supernatant to pass through a nickel column, and eluting by using eluents containing imidazole with different concentrations, wherein the concentration of the imidazole in the eluents is 20 mM-500 mM;
D. placing the enzyme solution obtained by 500mM elution in an ultrafiltration tube, centrifuging at the temperature of 4-16 ℃ and the rpm of 5000-6000, concentrating the protein, and adding a buffer solution B to suspend the protein in the concentration process. Buffer B consisted of the following components: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200ml of glycerol, 0.154 g of dithiothreitol, and the pH was adjusted to 7.9 with hydrochloric acid. Subpackaging the concentrated protein solution, and freezing and storing in a refrigerator at-80 ℃.
Preferably, the double enzyme couplingThe glycine transaminase in the method for detecting glutamic acid adopts the following coding gene sequences: first, with glycine transaminase AT 2The homology similarity of the coding gene sequence is more than 50%, and the coding protein has glycine transaminase activity; second, with glycine transaminase AT 2The amino acid sequence of the protein has more than 40% of consistency, and the encoded protein has glycine transaminase activity.
Preferably, the 3-phosphoglycerate dehydrogenase in the method for detecting glutamic acid by using the double enzyme coupling method adopts the following coding gene sequences: firstly, the homology similarity of the coding gene sequence and the sequence of the 3-phosphoglycerate dehydrogenase serA is more than 50 percent, and the coding protein has the gene sequence of the 3-phosphoglycerate dehydrogenase activity; second, the amino acid sequence identity to the 3-phosphoglycerate dehydrogenase serA protein is greater than 40%, and the encoded protein has 3-phosphoglycerate dehydrogenase activity.
The invention also comprises the application of the method for detecting the content of the glutamic acid by the enzyme method, which is used for developing a glutamic acid detection kit. For example, AT prepared according to the procedure of the present invention is determined according to the principle of the present invention for measuring glutamic acid content by the double enzyme coupling method 2serA, together with 100 mM TEA-HCl solution (pH 9.0), 100 mM sodium glyoxylate solution, 5 mM NADH solution, etc. were placed in a kit, and the kit was easily developed by making instructions for use according to the procedure of the present invention.
FIG. 1 is a schematic diagram showing the principle of the enzymatic method for detecting glutamic acid content in glutamic acid, glutamic acid and glyoxylic acid AT glycine transaminase (AT) 2) Is converted into glycine and 2-ketoglutaric acid; 3-phosphoglycerate dehydrogenase (serA) catalyzes the reduction of 2-ketoglutarate to 2-hydroxyglutarate, while NADH is oxidized to NAD +So that the absorbance of the system at 340nm is reduced; the detection method of the present invention requires the preparation of AT in addition to glyoxylic acid and NADH by commercial purchase 2And serA, determining AT according to literature reports by combining sequence alignment analysis means 2And serA coding gene, obtaining target gene by whole gene synthesis or PCR cloning, connecting expression vector, and transferring into special-purposeIn the escherichia coli or other expression hosts for protein expression, the purpose of large-scale exogenous expression, separation and purification of the target protein is realized;
second construction of AT-containing 2A reaction system of serA, glyoxylic acid, glutamic acid and NADH; optimizing the influence of factors such as buffer solution, pH, temperature, concentration and proportion of each enzyme and each reactant on the change of the absorbance of the system, estimating the detection limit and the quantification limit, determining the appropriate glutamic acid detection range, and making a standard curve;
finally, collecting clinical samples (including body fluids such as blood, urine and the like or tissues and the like) or preparing samples from general biological samples, and detecting the content of glutamic acid by using the detection system; it should be noted that glutamic acid has relevance to diseases such as diabetes, brain diseases and the like, and it is necessary to detect the value, but the value is only an intermediate value, and a certain disease cannot be diagnosed, and the diagnosis needs to be checked by a corresponding professional department.
Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:
the method for detecting the content of the glutamic acid by the enzyme method overcomes the defect that the traditional glutamate dehydrogenase method is not suitable for high-concentration ammonia environment, and creatively uses glycine transaminase AT 2The invention is coupled with 3-phosphoglycerate dehydrogenase serA double enzyme, deaminates the glutamic acid to form α -ketoglutaric acid, and rapidly and correctly characterizes the glutamic acid content in the system by reducing the NADH content through the reduction reaction of α -ketoglutaric acid.
The method for detecting the content of the glutamic acid by the enzyme method can realize high flux based on a microporous plate, can realize automatic operation by combining a liquid treatment workstation, further reduces the detection cost, can be used as a matching reagent for automatic analysis in the later period, and has good market prospect.
Drawings
FIG. 1 is a schematic diagram of the principle of the method for detecting the content of glutamic acid by an enzymatic method;
FIG. 2 is a standard curve of 0-100. mu.M glutamic acid concentration obtained in example 3 prepared by the endpoint method;
FIG. 3 is a calibration curve obtained by the initial velocity method for 0-100. mu.M glutamic acid concentration in example 3.
Detailed Description
The present invention will be further illustrated with reference to the following specific embodiments.
A method for detecting the content of glutamic acid by an enzyme method comprises the following steps:
exogenous expression and separation and purification of protein:
(1) selecting glycine transaminase AT by combining sequence alignment analysis means 2Then respectively synthesizing target gene by using whole gene and PCR to obtain target gene (in which the serA gene clone template is the genome of Escherichia coli E. coli k-12 strain), respectively obtaining glycine transaminase AT 2A target gene and a 3-phosphoglycerate dehydrogenase serA target gene;
(2) subjecting the glycine transaminase AT obtained in step (1) to 2The target gene and the 3-phosphoglycerate dehydrogenase serA target gene are respectively connected into a pET28a expression vector by molecular cloning, and pET28a-AT with correct sequencing after cloning is carried out 2After being transferred into the Escherichia coli Rosetta (DE3) strain together with pET28a-serA plasmid, the strain was cultured overnight at 37 ℃ in a test tube containing Luria-Bertani (LB) medium, and then transferred into a flask containing LB medium to OD 220 rpm at 37 ℃ in order to obtain a mixture 600AT about 0.6, IPTG was added to a final concentration of 1mM, and the expression was induced AT 37 ℃ for 4 hours to obtain glycine transaminase AT 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
(3) separating and purifying the target protein:
A. and (3) centrifuging the bacterial liquid containing the target protein at low temperature, removing supernatant, collecting thalli, adding buffer solution A, and suspending the protein in the buffer solution A. The centrifugation condition is 4-16 ℃, and the rotating speed is 10000-12000 rpm; buffer a consisted of the following components: 1L of buffer A contained 2.42 g of tris (hydroxymethyl) aminomethane, 37.3 g of potassium chloride, and 100 ml of glycerolThe pH was adjusted to 7.9 using hydrochloric acid. The target protein is glycine transaminase AT 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
B. after the cells are crushed by using a high-pressure cell crusher, centrifuging for 30-50 min at 4-16 ℃ and 10000-12000 rpm, and collecting supernatant;
C. enabling the supernatant to pass through a nickel column, and eluting by using eluents containing imidazole with different concentrations, wherein the concentration of the imidazole in the eluents is 20 mM-500 mM;
D. placing the enzyme solution obtained by 500mM elution in an ultrafiltration tube, centrifuging at the temperature of 4-16 ℃ and the rpm of 5000-6000, concentrating the protein, and adding a buffer solution B to suspend the protein in the concentration process. Buffer B consisted of the following components: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200ml of glycerol, 0.154 g of dithiothreitol, and the pH was adjusted to 7.9 with hydrochloric acid. Subpackaging the concentrated protein solution, and freezing and storing in a refrigerator at-80 ℃.
(II) detecting glutamic acid by a double-enzyme coupling method:
(1) preparing a reaction mixed solution:
preparing 100 mM TEA-HCl solution (pH 9.0), 100 mM sodium glyoxylate solution and 5 mM NADH solution for later use;
configuration of an AT-containing Glycine transaminase 2500. mu.l of a mixture of 3-phosphoglycerate dehydrogenase serA and the purified AT AT 3.4 mg/ml, 100. mu.l 2Adding the target protein and 80 mu l of the purified serA target protein with the concentration of 32 mg/ml, and then adding 320 mu l of buffer solution B to make up to 500 mu l for later use;
preparing reaction mixed liquor according to the solution, wherein the proportion of each 75 mul of reaction mixed liquor comprises: TEA-HCl solution, 25. mu.l; 10-30 mul of sodium glyoxylate aqueous solution; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; make up to 75. mu.l of ultrapure water.
(2) Preparing a glutamic acid concentration standard curve by an end-point method:
preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mu l of each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, gently mixing the glutamic acid standard solutions, and reacting for 15-30 minutes at the temperature of 30-40 ℃. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; 4-5 mul of NADH solution; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings. Drawing a glutamic acid concentration standard curve by taking the absorbance change value as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate;
(3) initial velocity method for preparing standard curve of glutamic acid concentration
Preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mu l of each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, gently mixing the glutamic acid standard solutions, and reacting for 2-10 minutes at the temperature of 30-40 ℃. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5-10 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader. And drawing a glutamic acid concentration standard curve by taking the absorbance change value at 340nm within 2-10 minutes as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate.
The operation steps of the invention are that the target gene of the whole gene synthesis is entrusted to the biological company for synthesis, and the step of the embodiment is synthesized by Nanjing Kingsler Biotech limited;
sodium glyoxylate, English name Sodium glyoxylate monohydrate, CAS number 918149-31-2;
the English name of NADH is Nicotinamide adenine dinucleotide with CAS number 74927-11-0;
the deproteinization kit adopted by the invention is Biovision.
The invention is further described with reference to specific examples.
Example 1
A method for detecting the content of glutamic acid by an enzyme method comprises the following steps:
exogenous expression and separation and purification of protein:
(1) selecting glycine transaminase AT by combining sequence alignment analysis means 2Then respectively synthesizing target gene by using whole gene and PCR to obtain target gene (in which the serA gene clone template is the genome of Escherichia coli E. coli k-12 strain), respectively obtaining glycine transaminase AT 2A target gene and a 3-phosphoglycerate dehydrogenase serA target gene;
according to the document "Masafumi Kameya, et al (2010) Purification of microorganisms from Hydrogenobacter thermophilusTK-6-novel types of ofalkane or a glycerol aminotransferase, FEBS Journal, 277:1876- Hydrogenobacter thermophilusGlycine aminotransferase AT is present in the TK-6 strain 2(amnT 2), searching the Gene database at NCBI for amnT2 to obtain the Gene sequence;
according to the document "Genshi ZHao, et al (1996) A novel α -ketoglutate reducation of the sera-encoded 3-phosphoglycerate dehydrogenase of Escherichia coliK-12 and its applicable identities for human 2-hydroxyglutamic acid. journal of Bacteriology, 178(1):232- Escherichia coli3-phosphoglycerate dehydrogenase SerA (NP-311811.1) exists in the K-12 strain, and a Gene sequence can be obtained by searching NP-311811.1 in a Gene database of NCBI;
glycine aminotransferase AT 2The target gene was synthesized by whole gene and assigned to the organism company, and 3-phosphoglycerate dehydrogenase serA was passed through Escherichia coliCarrying out PCR by taking the K-12 genome as a template to obtain a target gene;
wherein, AT 2The exogenous expression, separation and purification of the protein comprise the following specific steps:
（1）Masafumi Kameya, et al. (2010) Purification of three aminotransferasesfrom Hydrogenobacter thermophilusTK-6 – novel types of alanine or glycineaminotransferase. FEBS Journal, 277:1876-1885.
the above document reports Hydrogenobacter thermophilusGlycine aminotransferase AT is present in the TK-6 strain 2(amnT 2), searching the Gene database at NCBI for amnT2 to obtain the Gene sequence;
(2) sending the amnT2 sequence to Nanjing Kingsrei Biotech Co., Ltd for gene synthesis; enzyme cutting sites are designed at both ends EcoRI and HindIII；
(3) by using EcoRI and Hinenzyme digestion of the synthesized amnT2 by dIII, cutting of the gel, and recovery of gene fragments by using a gel recovery kit; concentration was measured using Nanodrop 2000;
(4) by using EcoRI and Hincarrying out enzyme digestion on the pET28a vector by dIII, cutting glue, and recovering a vector fragment by adopting a glue recovery kit; concentration was measured using Nanodrop 2000;
(5) constructing a ligation reaction system by using Solution I (ligase) to fragment in the steps 3 and 4, and ligating for 2 h at 16 ℃;
(6) conventional methods chemistry the ligation products were transformed into Rosetta (DE3) competent cells, plated on LB plates containing kanamycin;
(7) selecting 5 monoclonals to an LB culture medium, and after overnight culture, extracting plasmids by using a plasmid small quantity extraction kit;
(8) extracting the plasmid EcoRI and Hincarrying out enzyme digestion by dIII, and determining correct cloning by gel electrophoresis according to the size of the fragment;
(9) sequencing to verify the correctness of the correct clone in the step 8; the strain was stored in 10% glycerol.
(10) Inoculating the correct clone determined in step 9 into LB bottle, adding 1mM isopropyl- β -D-thiogalactoside with final concentration at OD600 = 0.6 to induce protein expression, and inducing at 37 deg.C and 200 rpm for 4 h;
(11) centrifuging the bacterial solution at 4 deg.C and 12000rpm for 5 min, removing supernatant, collecting thallus, adding buffer solution A, and suspending thallus in buffer solution A (buffer solution A comprises 1L buffer solution A containing 2.42 g of tris (hydroxymethyl) aminomethane, 37.3 g of potassium chloride, 100 ml of glycerol, and adjusting pH to 7.9 with hydrochloric acid);
(12) after the cells are crushed by a high-pressure cell crusher, centrifuging for 30 min at the temperature of 4 ℃ and the rpm of 11000, and collecting supernatant;
(13) the supernatant is passed through a nickel column and the protein is purified based on the principle of affinity chromatography. Eluting by using eluents containing imidazole with different concentrations, wherein the concentration of the imidazole in the eluents is 20 mM-500 mM;
(14) the 500mM eluted enzyme solution was placed in an ultrafiltration tube, protein concentration was performed at 4 ℃ and 5000 rpm, and buffer B was added during concentration to suspend the protein. Buffer B consisted of the following components: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200ml of glycerol, 0.154 g of dithiothreitol, and the pH was adjusted to 7.9 with hydrochloric acid. . Subpackaging the concentrated protein solution, and freezing and storing in a refrigerator at-80 ℃.
Wherein, the exogenous expression, separation and purification of the serA protein comprise the following specific steps:
（1）Genshi Zhao, et al. (1996) A novel α-ketoglutarate reductase activityof the sera-encoded 3-phosphoglycerate dehydrogenase of Escherichia coliK-12and its possible implications for human 2-hydroxyglutaric aciduria. Journalof Bacteriology, 178(1):232-239.
the above document reports Escherichia coliThe K-12 strain contains 3-phosphoglycerate dehydrogenase SerA (NP-311811.1), and the Gene sequence can be obtained by searching NP-311811.1 in the Gene database of NCBI.
(2) Designing according to the obtained gene sequence serAThe primers of the gene are synthesized by the Nanjing Jinslei Biotech limited company by designing enzyme cutting sites SacI and NotI at two ends Escherichia coliPerforming PCR by using KOD polymerase with K-12 genome as a template;
(3) performing DNA electrophoresis on a product obtained by PCR, cutting gel, and recovering a gene fragment by using a gel recovery kit; concentration was measured using Nanodrop 2000;
(4) by using SacI and Noti enzyme digestion of the serA obtained by the PCR, gel cutting, recovery by a gel recovery kitA gene fragment; concentration was measured using Nanodrop 2000;
(5) by using SacI and Noti, carrying out enzyme digestion on a pET28a vector, cutting glue, and recovering a vector fragment by adopting a glue recovery kit; concentration was measured using Nanodrop 2000;
(6) constructing a ligation reaction system by utilizing Solution I (ligase) to fragment in the steps 4 and 5, and ligating for 2 h at 16 ℃;
(7) conventional methods chemistry the ligation products were transformed into Rosetta (DE3) competent cells, plated on LB plates containing kanamycin;
(8) 3 monoclonals are selected to be placed in an LB culture medium, and after overnight culture, plasmids are extracted by a plasmid small quantity extraction kit;
(9) extracting the plasmid SacI and Noti, enzyme digestion, and determining correct clone by gel electrophoresis according to the size of a fragment;
(10) sequencing to verify the correctness of the correct clone in the step 9; the strain was stored in 10% glycerol.
(11) Inoculating the correct clone determined in step 10 into LB bottle, adding 1mM isopropyl- β -D-thiogalactoside at final concentration when OD600 is 0.6 to induce protein expression, and inducing at 37 deg.C and 200 rpm for 4 h;
(12) centrifuging the bacterial solution at 4 deg.C and 12000rpm for 5 min, removing supernatant, collecting thallus, adding buffer solution A, and suspending thallus in buffer solution A (buffer solution A comprises 1L buffer solution A containing 2.42 g of tris (hydroxymethyl) aminomethane, 37.3 g of potassium chloride, 100 ml of glycerol, and adjusting pH to 7.9 with hydrochloric acid);
(13) after the cells are crushed by a high-pressure cell crusher, centrifuging for 50 min at the temperature of 4 ℃ and the rpm of 11000, and collecting supernatant;
(14) the supernatant is passed through a nickel column and the protein is purified based on the principle of affinity chromatography. Eluting by using eluents containing imidazole with different concentrations, wherein the concentration of the imidazole in the eluents is 20 mM-500 mM;
(15) the 500mM eluted enzyme solution was placed in an ultrafiltration tube, protein concentration was performed at 4 ℃ and 5000 rpm, and buffer B was added during concentration to suspend the protein. Buffer B consisted of the following components: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200ml of glycerol, 0.154 g of dithiothreitol, and the pH was adjusted to 7.9 with hydrochloric acid. Subpackaging the concentrated protein solution, and freezing and storing in a refrigerator at-80 ℃.
(II) detecting glutamic acid by a double-enzyme coupling method:
(1) preparing a reaction mixed solution:
preparing 100 mM TEA-HCl solution (pH 9.0), 100 mM sodium glyoxylate solution and 5 mM NADH solution for later use; configuration of an AT-containing Glycine transaminase 2500. mu.l of a mixture of 3-phosphoglycerate dehydrogenase serA and purified AT AT 3.4 mg/ml in 100. mu.l 2Adding the target protein and 80 mu l of the purified serA target protein with the concentration of 32 mg/ml, and then adding 320 mu l of buffer solution B to make up to 500 mu l for later use;
preparing reaction mixed liquor according to the solution, wherein the proportion of each 75 mul of reaction mixed liquor comprises: TEA-HCl solution, 25. mu.l; sodium glyoxylate aqueous solution, 30. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; make up to 75. mu.l of ultrapure water.
(2) Preparing a glutamic acid concentration standard curve:
end-point method: preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mu l of each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, gently mixing the glutamic acid standard solutions, reacting at 37 ℃ for 20 minutes, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings. Drawing a glutamic acid concentration standard curve by taking the absorbance change value as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate;
initial velocity method: preparing a series of glutamic acid standard solutions with the concentration of 0-0.4 mM, respectively taking 25 mu l of each concentration, respectively dripping the glutamic acid standard solutions into a 96-hole enzyme label plate filled with 75 mu l of reaction mixed solution, gently mixing the glutamic acid standard solutions, and reacting for 5 minutes at 37 ℃. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader. And drawing a glutamic acid concentration standard curve by taking the absorbance change value at 340nm in 5 minutes as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate.
(III) detection of glutamic acid in urine, blood, or general biological samples:
(1) urine treatment and detection
End-point method: treating urine by using a deproteinization kit, centrifuging to obtain supernatant, and obtaining a urine sample; during detection, 25 mul of urine is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed solution, the mixture is mixed evenly and lightly, and the reaction is finished after 20 minutes of reaction at 37 ℃. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; urine, 25 μ l; make up to 100. mu.l of ultrapure water. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings.
And calculating the glutamic acid content of the urine according to the change value of the absorbance of the urine sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: treating urine by using a deproteinization kit, centrifuging to obtain supernatant, and obtaining a urine sample; when in detection, 25 mul urine is added into a 96-hole enzyme label plate filled with 75 mul reaction mixed solution, the mixture is mixed lightly and reacts for 5 minutes at 37 ℃, and the proportion of the reaction mixed solution of a blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the urine according to the change value of the absorbance of the urine sample within 5 minutes and a glutamic acid standard curve prepared by an initial velocity method.
(2) Blood treatment and detection
The blood sample used in the present invention is stored in an anticoagulation tube, so that the blood sample contains the anticoagulant of the blood sample, and the anticoagulant in the anticoagulation tube is 10% potassium oxalate-sodium fluoride.
When a blood sample is detected, the blood sample needs to be pretreated, and the steps are as follows: the blood in the anticoagulation tube needs to be firstly centrifuged at low speed to obtain plasma under the condition of 4 ℃ and 4000 rpm for 10 min, and then centrifuged at high speed to remove impurities which can interfere with the experiment from the plasma under the condition of 4 ℃ and 14000 rpm for 10 min. And (4) centrifuging, and taking the supernatant to measure the content of glutamic acid.
End-point method: separating blood to obtain plasma or serum to obtain blood sample; when in detection, 25 mul of blood plasma or blood serum is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed liquid, the mixture is mixed lightly and evenly, the reaction is finished after 20 minutes of reaction at 37 ℃, and the proportion of the reaction mixed liquid of blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; plasma or serum, 25 μ l; make up to 100. mu.l of ultrapure water. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings.
And calculating the glutamic acid content of the blood according to the change value of the absorbance of the plasma or serum sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: separating blood to obtain plasma or serum to obtain blood sample; when in detection, 25 mul of blood plasma or blood serum is added into a 96-hole enzyme label plate filled with 75 mul of reaction mixed liquid, the mixture is mixed evenly and reacts for 5 minutes at 37 ℃, and the proportion of the reaction mixed liquid of a blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the plasma or serum sample according to the change value of the absorbance of the plasma or serum sample within 5 minutes and a glutamic acid standard curve prepared by an initial velocity method.
(3) Detection of biological samples in general
End-point method: after a general biological sample is treated, taking supernatant, adding 25 mul of biological sample into a 96-hole enzyme label plate filled with 75 mul, mixing the biological sample and the enzyme label plate gently, reacting for 20 minutes at 37 ℃, and finishing the reaction, wherein the reaction mixed solution of a blank control comprises the following components in proportion: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; general biological sample, 25. mu.l; make up to 100. mu.l of ultrapure water. And (3) reading the absorbance at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original absorbance reading and the rest readings.
And calculating the glutamic acid content according to the change value of the absorbance of the general biological sample and a glutamic acid standard curve made by an end-point method.
Initial velocity method: after a general biological sample is treated, supernatant is taken, 25 mul of biological sample is added into a 96-hole enzyme label plate filled with 75 mul, the biological sample is mixed evenly and lightly and reacts for 5 minutes at 37 ℃, and the proportion of the reaction mixed solution of a blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.
And calculating the glutamic acid content of the plasma or serum sample according to the change value of the absorbance of the general biological sample within 5 minutes and a glutamic acid standard curve prepared by an initial velocity method.
Example 2
The conditions were the same as in example 1 except for the proportional composition of 75. mu.l of the mixture;
the composition of the reaction mixture per 75. mu.l of reaction mixture was: TEA-HCl solution, 25. mu.l; 20 mul of sodium glyoxylate aqueous solution; NADH solution, 4. mu.l; 10 mul of double-enzyme mixed solution; ultrapure water, 16. mu.l.
Example 3
The conditions were the same as in example 1 except for the proportional composition of 75. mu.l of the mixture;
the composition of the reaction mixture per 75. mu.l of reaction mixture was: TEA-HCl solution, 25. mu.l; 10 μ l of aqueous sodium glyoxylate solution; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; ultrapure water, 30. mu.l.
After an enzyme-labeling instrument is used for detecting an absorbance value at 340nm by an end-point method or an initial velocity method, a glutamic acid concentration standard curve is drawn by taking an absorbance change value as a vertical coordinate and a glutamic acid standard solution concentration as a horizontal coordinate, and a standard curve is drawn by software such as Excel and the like, so that the concentration of 0-100 mu m of glutamic acid has a good linear relation, and therefore, the concentration of 0-100 mu m of glutamic acid is selected as a main measurement range, the standard curve is shown in figure 2 and is made by the end-point method under the concentration of 0-100 mu m of glutamic acid, and the standard curve is shown in figure 3 and is made by the initial velocity method under the concentration of 0-100 mu m of glutamic acid; therefore, when the glutamic acid concentration in the sample is more than this range, the sample is appropriately diluted.
The plasma glutamic acid concentration was measured using the standard curve obtained in example 3 at a glutamic acid concentration of 0 to 100. mu.M, and the following procedure was carried out:
(1) collecting peripheral blood of 42 conventional physical examination personnel according to the requirements of ethical specifications of hospitals;
(2) separating with plasma separating hose to obtain plasma, and storing at-80 deg.C;
(3) during the assay, 25. mu.l of the supernatant from step 2 was added to a 96-well plate (CLS 3590-100EA, Corning), and then 75. mu.l of the reaction mixture (example 3) was added and gently mixed;
(4) end-point method: the reaction is finished after 20 minutes of reaction at 37 ℃, and the proportion of the reaction mixed liquor of the blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 25. mu.l; NADH solution, 5. mu.l; 5 mul of double-enzyme mixed solution; plasma, 25 μ l, make up ultrapure water to 100 μ l. And after the reaction is finished, reading the light absorption value at 340nm by using a microplate reader, wherein the change value of the absorbance is the difference between the original light absorption value reading and the rest readings.
Initial velocity method: reacting at 37 ℃ for 5 minutes, wherein the proportion of the reaction mixture of the blank control comprises: TEA-HCl solution, 25. mu.l; 5 mul of double-enzyme mixed solution; make up to 100. mu.l of ultrapure water. And monitoring the change of the absorbance at 340nm by using a microplate reader to obtain the change of the absorbance at 340nm within 5 minutes.
(5) According to the change value of the absorbance of the blood sample and a glutamic acid standard curve made by an end-point method or an initial velocity method, the glutamic acid content of the blood can be calculated, and the average glutamic acid concentration value of the part of people is 125.1 +/-57.5 mu M.
The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Claims (10)

1. A method for detecting the content of glutamic acid by an enzyme method is characterized by comprising the following steps: using glycine transaminase AT 2And a two-enzyme coupling system of 3-phosphoglycerate dehydrogenase SerA, and determining the content of glutamic acid.
2. The method for detecting the content of the glutamic acid by the enzyme method according to claim 1, which comprises the following steps:
(1) exogenous expression and separation and purification of protein
1) Preparation of Glycine transaminase AT 2A target gene and a 3-phosphoglycerate dehydrogenase serA target gene;
2) subjecting the glycine transaminase AT obtained in step 1) 2The target gene and the 3-phosphoglycerate dehydrogenase serA target gene are respectively connected into a pET28a expression vector to obtain pET28a-AT 2And pET28a-serA plasmid;
3) pET28a-AT 2Respectively transferring the plasmid and pET28a-serA into an escherichia coli Rosetta strain, and culturing in a culture medium to obtain the sweetAlanine aminotransferase AT 2A target protein and a 3-phosphoglycerate dehydrogenase serA target protein;
4) separating and purifying the target protein;
(2) and (3) detecting the content of glutamic acid in the sample by using the target protein obtained by separation and purification through a double-enzyme coupling method.
3. The method for detecting the content of glutamic acid by the enzymatic method according to claim 2, wherein the step of separating and purifying the target protein in the step 4) comprises the following steps:
A. the glycine-containing transaminase AT obtained in the step 3) is treated 2Centrifuging the bacterial liquid of the target protein and the 3-phosphoglycerate dehydrogenase SerA target protein at low temperature, removing supernatant, collecting thalli, adding a buffer solution A, and suspending the thalli in the buffer solution A;
B. crushing the thallus, centrifuging and collecting supernatant;
C. passing the supernatant through a nickel column, and eluting with an eluent;
D. and placing the enzyme solution obtained by elution in an ultrafiltration tube for centrifugation and concentration, and subpackaging the concentrated protein solution to obtain the separated and purified target protein.
4. The method for detecting the content of glutamic acid by the enzymatic method according to claim 3, wherein in the step A, the reaction temperature of centrifugation is 4-16 ℃, and the rotation speed is 10000-12000 rpm; in the step D, the reaction temperature of centrifugation is 4-16 ℃, and the rotating speed is 5000-6000 rpm.
5. The method for detecting the content of glutamic acid by the enzymatic method according to claim 3, wherein in the step A, the buffer A consists of the following components: 1L of buffer A contained 2.42 g of tris, 37.3 g of potassium chloride and 100 ml of glycerol; in the step D, adding a buffer solution B in the concentration process, and suspending the protein in the buffer solution B; buffer B consisted of the following components: 1L of buffer B contained 2.42 g of Tris, 7.45 g of potassium chloride, 200ml of glycerol and 0.154 g of dithiothreitol.
6. The method for detecting the content of glutamic acid by the enzymatic method according to claim 3, wherein in the step C, an eluent contains imidazole, and the concentration of the imidazole in the eluent is 20-500 mmol/L.
7. The method for detecting the content of glutamic acid by the enzymatic method according to claim 2, wherein: the double-enzyme coupling method in the step (2) is an end-point method or an initial velocity method.
8. The method for detecting the content of glutamic acid by the enzymatic method according to claim 7, wherein the detection steps of the endpoint method are as follows: preparing a glutamic acid standard solution with the concentration of 0-0.4 mmol/L, respectively dripping 25 mul of glutamic acid standard solution with each concentration into a 96-hole enzyme label plate filled with 75 mul of reaction mixed solution, uniformly mixing, reacting for 15-30 minutes at 30-40 ℃, finishing the reaction, reading the light absorption value at 340nm by using an enzyme label reader, and drawing a glutamic acid concentration standard curve by taking the variation value of the light absorption as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate.
9. The method for detecting the content of glutamic acid by the enzymatic method according to claim 7, wherein: the initial velocity method comprises the following detection steps: preparing a glutamic acid standard solution with the concentration of 0-0.4 mmol/L, respectively dripping 25 mul of glutamic acid standard solution with each concentration into a 96-hole enzyme label plate filled with 75 mul of reaction mixed solution, uniformly mixing, reacting for 2-10 minutes at 30-40 ℃, monitoring the variation of absorbance at 340nm in the reaction process by using an enzyme label reader, and drawing a glutamic acid concentration standard curve by taking the variation of absorbance at 340nm in 2-10 minutes as a vertical coordinate and the concentration of the glutamic acid standard solution as a horizontal coordinate.
10. The method for detecting the content of glutamic acid by the enzymatic method according to claim 8 or 9, wherein: the composition of the reaction mixture per 75. mu.l of reaction mixture was: TEA-HCl solution, 25. mu.l; 10-30 mul of sodium glyoxylate aqueous solution; 4-5 mul of NADH solution; 5-10 mul of the double-enzyme mixed solution, and supplementing ultrapure water to 75 mul.
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