阅读说明：本技术 一种酶法检测甘氨酸含量的方法及其应用 (Method for detecting glycine content by enzyme method and application thereof ) 是由 朱虹 花强 董辉 金维荣 邓伟伟 于 2019-10-31 设计创作，主要内容包括：本发明涉及一种酶法检测甘氨酸含量的方法及其应用,利用甘氨酸氧化酶Glycine Oxidase以及乙醛酸还原酶Glyoxylate Reductase的双酶偶联体系,测定甘氨酸的含量；本发明的酶法检测甘氨酸含量的方法克服了传统甘氨酸氧化酶偶联辣根过氧化物酶HRP方法不适用于血液环境的缺陷,创造性地通过甘氨酸氧化酶Glycine Oxidase以及乙醛酸还原酶Glyoxylate Reductase双酶偶联的方法,将甘氨酸氧化形成乙醛酸,又通过乙醛酸的还原反应,以NADH含量的减少来快速正确表征体系中的甘氨酸含量；本发明灵敏度高,检测迅速稳定,重复性好,操作简便,成本较低,适用范围较广。(The invention relates to a method for detecting Glycine content by an enzyme method and application thereof, which utilizes a double-enzyme coupling system of Glycine Oxidase Glycine Oxidase and Glyoxylate Reductase Glycyxylate Reductase to determine the content of Glycine; the method for detecting the Glycine content by the enzyme method overcomes the defect that the traditional method for coupling Glycine Oxidase with horseradish peroxidase (HRP) is not suitable for blood environment, creatively oxidizes the Glycine to form glyoxylic acid by a double-enzyme coupling method of Glycine Oxidase Glycine Oxidase and glyoxylic acid Reductase Glycoxylate Reductase, and rapidly and correctly represents the Glycine content in a system by reducing the NADH content through the reduction reaction of the glyoxylic acid; the invention has the advantages of high sensitivity, rapid and stable detection, good repeatability, simple operation, low cost and wide application range.)

1. A method for detecting glycine content by an enzymatic method is characterized by comprising the following steps: the content of Glycine is determined by using a double-enzyme coupling system of Glycine Oxidase and Glyoxylate Reductase.

2. The method for detecting the content of the glycine by the enzymatic method according to claim 1, characterized by comprising the following steps:

(1) exogenous expression and separation and purification of protein

1) Preparing a Glycine Oxidase Glycine Oxidase target gene and a glyoxylate reductase GlycyxylateReductase target gene;

2) respectively connecting the Glycine Oxidase glucose Oxidase target gene and the Glyoxylate Reductase glyxoylate Reductase target gene obtained in the step 1) into a pET28a expression vector to obtain pET28a-GO and pET28a-GR plasmids;

3) respectively transferring pET28a-GO and pET28a-GR plasmids into an escherichia coli Rosetta strain, and culturing in a culture medium to obtain a Glycine Oxidase target gene and a glyoxylate reductase glyxylateReductase target protein;

4) separating and purifying the target protein;

(2) and (3) detecting the content of the glycine in the sample by using the target protein obtained by separation and purification through a double-enzyme coupling method.

3. The method for detecting glycine content by using 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. centrifuging the bacterial liquid containing the Glycine Oxidase target gene and the Glyoxylate Reductase glyoxalate Reductase target protein obtained in the step 3) 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 glycine content by using the enzymatic method according to claim 3, wherein the enzymatic method comprises the following steps: in the step A, the reaction temperature of centrifugation is 4-16 ℃, and the rotating 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 glycine content by using the enzymatic method according to claim 3, wherein in the step A, the buffer A consists of the following components: 1L of buffer A contains 2.42 g of tris (hydroxymethyl) aminomethane, 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; wherein the buffer B consists of the following components: 1L of buffer B contained 2.42 g of tris, 7.45 g of potassium chloride, 200 ml of glycerol and 0.154 g of dithiothreitol.

6. The method for detecting glycine content by using the enzymatic method according to claim 3, wherein the enzymatic method comprises the following steps: in the step C, the 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 the glycine by the enzymatic method according to claim 2, wherein the enzymatic method comprises the following steps: the double-enzyme coupling method in the step (2) is an end-point method or an initial velocity method.

8. The method for detecting glycine content by using the enzymatic method according to claim 7, wherein the detection steps of the endpoint method are as follows: preparing glycine standard solution with concentration of 0-1.2 mmol/L, respectively dripping 25 mul of glycine standard solution with each concentration into a 96-hole enzyme label plate filled with 175 mul of reaction mixed solution, uniformly mixing, reacting for 20-40 minutes at 30-40 ℃, finishing the reaction, reading the light absorption value at 340nm by using an enzyme label instrument, and drawing a glycine concentration standard curve by taking the variation value of the light absorption as ordinate and the concentration of the glycine standard solution as abscissa.

9. The method for detecting glycine content by using the enzymatic method according to claim 7, wherein the detection steps of the initial velocity method are as follows: preparing glycine standard solution with the concentration of 0-1.2 mmol/L, respectively dripping 25 mul of glycine standard solution with each concentration into a 96-hole enzyme label plate filled with 175 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 instrument, and drawing a glycine concentration standard curve by taking the variation of absorbance at 340nm in 2-10 minutes as a vertical coordinate and the concentration of the glycine standard solution as a horizontal coordinate.

10. The method according to claim 8 or 9, wherein the proportion of reaction mixture per 175 μ l comprises: 100 mul of TEA-HCl solution, 10-30 mul of flavin adenine dinucleotide disodium water solution, 8-10 mul of NADH solution, 10-15 mul of double-enzyme mixed solution, and supplementing ultrapure water to 175 mul.

Technical Field

The invention relates to the technical field of enzymatic detection, in particular to a method for detecting glycine content by an enzymatic method and application thereof.

Background

The existing methods for detecting glycine 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 determination of complex samples, but the method for determining the glycine by the liquid chromatography needs to perform pre-column derivatization on the samples, and the instruments of the liquid chromatography and the ion chromatography are expensive, time-consuming and high in cost, and are not suitable for analysis and determination of high-throughput samples.

The detection system for determining the glycine content by the traditional spectrophotometry is mainly based on the coupling reaction of glycine oxidase GlycineOxidate and horseradish peroxidase HRP, and the concentration of glycine is determined by a chromogenic system of a product hydrogen peroxide-phenol-4-aminoantipyrine or a product hydrogen peroxide and OxiRed Probe. However, when the method is used for detecting a blood sample, the enzyme activity of HRP is inhibited by the blood environment, the reaction cannot be normally carried out, and an accurate result of the glycine content in the sample cannot be obtained. This limits the use of glycine oxidase-horseradish peroxidase and its kit in the above samples.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for detecting glycine content 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 Glycine content by an enzyme method utilizes a double-enzyme coupling system of Glycine Oxidase Glycine Oxidase and Glyoxylate Reductase Glycyxylate Reductase to realize the determination of the Glycine content;

wherein the nucleotide sequence of Glycine Oxidase Glycine Oxidase is as follows:

ATGAAAAGGCATTATGAAGCAGTGGTGATTGGAGGCGGAATTATCGGTTCCGCAATTGCTTATTATTTGGCAAAGGAAAACAAAAACACCGCATTGTTTGAAAGCGGAACAATGGGCGGCAGAACGACAAGTGCCGCTGCCGGAATGCTGGGCGCCCATGCCGAATGCGAGGAACGTGACGCGTTTTTTGATTTCGCCATGCACAGCCAGCGTCTGTACAAAGGTCTTGGAGAAGAGCTTTATGCATTATCCGGTGTGGATATCAGGCAGCATAACGGCGGTATGTTTAAACTTGCATTTTCTGAAGAAGATGTGCTGCAGCTGAGACAGATGGACGATTTGGACTCTGTCAGCTGGTATTCAAAAGAAGAGGTGTTAGAAAAAGAGCCGTATGCGTCTGGTGACATCTTTGGTGCATCTTTTATTCAGGATGATGTGCATGTGGAGCCTTATTTTGTTTGCAAGGCATATGTGAAAGCAGCAAAAATGCTTGGGGCGGAGATTTTTGAGCATACGCCCGTCCTGCATGTCGAACGTGACGGTGAAGCCCTGTCCATCAAGACCCCTAGCGGAGACGTATGGGCTAATCATGTTGTCGTTGCCAGCGGGGTGTGGAGCGGAATGTTTTTTAAACAGCTTGGACTGAACAATGCTTTTCTCCCTGTAAAAGGGGAGTGCCTGTCCGTTTGGAATGATGATATCCCGCTGACAAAAACGCTTTACCATGATCACTGCTATATCGTACCGAGAAAAAGCGGCAAACTGGTTGTCGGCGCGACAATGAAGCCGGGGGACTGGAGTGAAACACCGGATCTTGGCGGATTGGAATCGGTTATGAAAAAAGCAAAAACGATGCTGCCGGCTATACAGAATATGAAGGTGGATCGTTTTTGGGCGGGACTCCGGCCGGGAACAAAGGATGGAAAACCGTACATCGGCAGACATCCTGAGGACAGCCGTATTTTATTTGCGGCGGGCCATTTCAGAAATGGGATCCTGCTTGCTCCCGCAACGGGCGCTTTGATCAGTGATCTCATCATGAATAAAGAGGTCAACCAAGACTGGCTGCACGCATTCCGAATTGATCGCAAGGAGGCGGTTCAGATATGA；

the amino acid sequence of Glycine Oxidase Glycine Oxidase is:

MKRHYEAVVIGGGIIGSAIAYYLAKENKNTALFESGTMGGRTTSAAAGMLGAHAECEERDAFFDFAMHSQRLYKGLGEELYALSGVDIRQHNGGMFKLAFSEEDVLQLRQMDDLDSVSWYSKEEVLEKEPYASGDIFGASFIQDDVHVEPYFVCKAYVKAAKMLGAEIFEHTPVLHVERDGEALSIKTPSGDVWANHVVVASGVWSGMFFKQLGLNNAFLPVKGECLSVWNDDIPLTKTLYHDHCYIVPRKSGKLVVGATMKPGDWSETPDLGGLESVMKKAKTMLPAIQNMKVDRFWAGLRPGTKDGKPYIGRHPEDSRILFAAGHFRNGILLAPATGALISDLIMNKEVNQDWLHAFRIDRKEAVQI；

the nucleotide sequence of Glyoxylate Reductase glyyxylate Reductase is as follows:

TCACGGCGGGGAGGATGTCAGAACATCTTTGTTGACAAGGTTCGGTGGGATTTCACCTTTGGCAAATGCTATTAGGTTCTTTGCAACCAGCTCCGCCATGCCTTCTCGTGCTTCATGAGTTGCGCTGCCTATGTGGGGAGCTAAAACAACGTTCTTCAACTTGAAAAGCTCCTCGTTATAGTAGGGTTCTTCTTCAAATACATCCAAGCCTGCTCCAGCAATCCACCCTTCTTTTAATGCTTTGATTAGGGCATTAGTATCGACAACTGCCCCTCTAGATGTGTTTATTAGGATTGCGTTGGGTTTCATAAGCTTGAGCTCCTTCTCTCCTATCATGTGGTAGGTCTCTTTGGTTAGGGGAACGTGAAGGCTTATAAAGTCGCTCTCTTTCAAAAGGGTTTCAAAATCCACATACTCTGCGCCAATCTCTTCTTCTGCTTCAGGTTTACGTGTTCTTGAGTAATAGATGATTTTCATCCCGAACCCTTTGGCTCTCTTTGCGAGGGCCTGGCCGATTCTTCCAAAGCCCACTATCCCTAAAGTCTTTCCTTTCAATCCATATCCTAGAAACATTAGAGGATGCCAGCCGACTTCGCTTTTCTTCCATTCGCCACTCCTCACAAAGGCATCAGCCTCTACAATTCTCCTCGCAACTGCTAGAAGAAGGGCAAATGCAAGGTCAGCCGTTGCATCCGTAAGGACTCCGGGAGTGTTTGTAACGTATATTCCCCTTTTTGTAGCCTCTTCTATGTCTATGTTATCATAGCCGACTGCATATTGGGCTATTATTTTTAACTTTGGAGCGTTTTCCAGTAGTTCTTTATCTACTTTGTCCGTTACAAGGGTCACTAAGGCATCAACCTCTCTGACTTTCTCTAGAAGCACTCCCCGTGGAGGTGCCTTCGGATCTTTCCAGAGTTCTATTTCATAGAATTTCTCAATCATCTTAATTCCATTTTCCGGAATTTGTCGTGTTATAAACACCTTGGGCTTCAT；

the amino acid sequence of Glyoxylate Reductase glyyxylate Reductase is: MKPKVFITRQIPENGIKMIEKFYEIELWKDPKAPPRGVLLEKVREVDALVTLVTDKVDKELLENAPKLKIIAQYAVGYDNIDIEEATKRGIYVTNTPGVLTDATADLAFALLLAVARRIVEADAFVRSGEWKKSEVGWHPLMFLGYGLKGKTLGIVGFGRIGQALAKRAKGFGMKIIYYSRTRKPEAEEEIGAEYVDFETLLKESDFISLHVPLTKETYHMIGEKELKLMKPNAILINTSRGAVVDTNALIKALKEGWIAGAGLDVFEEEPYYNEELFKLKNVVLAPHIGSATHEAREGMAELVAKNLIAFAKGEIPPNLVNKDVLTSSPP are provided.

Preferably, the method for detecting the glycine content by the enzymatic method comprises the following steps:

exogenous expression and separation and purification of protein:

(1) combining a sequence comparison analysis means, selecting a gene coding sequence of Glycine Oxidase Glycine Oxidase and a gene coding sequence of Glyoxylate Reductase Glycosylate Reductase, and then obtaining a target gene of Glycine Oxidase Glycine Oxidase and a target gene of Glyoxylate Reductase Glycosylate Reductase by a method of synthesizing the target gene by whole genes;

(2) respectively connecting the Glycine Oxidase glucose Oxidase target gene and the Glyoxylate Reductase target gene obtained in the step (1) into a pET28a expression vector by adopting molecular cloning, respectively transferring pET28a-GO and pET28a-GR plasmids with correct sequencing after cloning into an escherichia coli Rosetta (DE3) strain, culturing the escherichia coli Rosetta (LB) strain in a test tube filled with Luria-Bertani (LB) culture medium at 37 ℃ for overnight,then transferred into a shake flask containing LB medium, cultured at 37 ℃ and 220 rpm to OD₆₀₀Adding IPTG with the final concentration of 1mM when the concentration is about 0.6, and carrying out induced expression for 12 hours at the temperature of 16 ℃ to respectively obtain Glycine Oxidase target protein and Glyoxylate Reductase glycoxylate Reductase target protein;

(3) separating and purifying the target protein: centrifuging a bacterium solution containing a target protein at low temperature to collect thalli, crushing the thalli, centrifuging to collect supernatant, and separating and purifying the target protein by adopting a nickel column for later use, wherein the target protein comprises the Glycine Oxidase glucose Oxidase target protein and the Glyoxylate Reductase glyxylate Reductase target protein obtained in the step (2);

(II) detecting glycine by a double-enzyme coupling method:

(1) preparing a reaction mixed solution:

preparing 100 mM TEA-HCl solution (pH 9.0), 100 mu M flavin adenine dinucleotide disodium water solution and 5 mM ADH solution for later use; 500 mul of double-enzyme mixed solution containing Glycine Oxidase Glycine Oxidase and glyoxylate reductase Glycosylate Reductase is prepared, and the mixed solution contains 416 mul of purified GO target protein with the concentration of 8.58mg/ml and 84 mul of purified GR target protein with the concentration of 8.49mg/ml for later use.

Preparing a reaction mixed solution according to the solution, wherein the proportion of each 175 mu l of the reaction mixed solution comprises the following components: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; 10-15 mul of double-enzyme mixed solution; make up to 175. mu.l of ultrapure water.

(2) The end-point method is used for preparing a glycine concentration standard curve:

preparing a series of glycine standard solutions with the concentration of 0-1.2 mM, respectively taking 25 mu l of glycine standard solution with each concentration, respectively dripping the glycine standard solutions into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, gently mixing the glycine standard solutions, reacting at the temperature of 30-40 ℃ for 20-40 minutes, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. mu.l. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. 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. Drawing a glycine concentration standard curve by taking the absorbance change value as a vertical coordinate and the concentration of the glycine standard solution as a horizontal coordinate;

(3) initial velocity method for preparing glycine concentration standard curve

Preparing a series of glycine standard solutions with the concentration of 0-1.2 mM, respectively taking 25 mu l of glycine standard solution with each concentration, respectively dripping the glycine standard solutions into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, gently mixing the glycine standard solutions, and reacting for 2-10 minutes at the temperature of 30-40 ℃. The blank control was ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader. And drawing a glycine concentration standard curve by taking the absorbance change value at 340nm within 2-10 minutes as a vertical coordinate and taking the concentration of the glycine standard solution as a horizontal coordinate.

(III) detection of Glycine in urine, blood, or in 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 175 mu l of reaction mixed solution, slightly and uniformly mixing, reacting for 20-40 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, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. mu.l. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; urine 25 μ l, make up ultrapure water to 200 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 glycine content of the urine according to the change value of the absorbance of the urine sample and a glycine standard curve prepared by an end-point method.

Initial velocity 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 175 mu l of reaction mixed liquid, slightly and uniformly mixing, reacting for 2-10 minutes at 30-40 ℃, and using a blank control as ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.

And calculating the glycine content of the urine according to the change value of the absorbance of the urine sample within 2-10 minutes and a glycine standard curve prepared by an initial speed 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 4000rpm 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 14000rpm for 10 min. And (4) centrifuging, and taking the supernatant to measure the glycine content.

End-point method: separating blood to obtain plasma or serum to obtain blood sample; and during detection, adding 25 mu l of plasma or serum into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, slightly and uniformly mixing, reacting for 20-40 minutes at 30-40 ℃, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. mu.l. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; plasma or serum 25 μ l, make up ultrapure water to 200 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 glycine content of the blood according to the change value of the absorbance of the plasma or serum sample and a glycine standard curve prepared by an end-point method.

Initial velocity method: separating blood to obtain plasma or serum to obtain blood sample; and during detection, adding 25 mu l of plasma or serum into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed liquid, slightly and uniformly mixing, reacting for 2-10 minutes at 30-40 ℃, and using a blank control as ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.

And calculating the glycine 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 glycine standard curve prepared by an initial speed method.

(3) Detection of biological samples in general

End-point method: and (3) treating a general biological sample, taking supernatant, adding 25 mu l of biological sample into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, slightly and uniformly mixing, reacting at 30-40 ℃ for 20-40 minutes, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. mu.l. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; typically 25. mu.l of biological sample is supplemented with ultrapure water to 200. 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 according to the change value of the absorbance of the general biological sample and a glycine standard curve made by an end-point method to obtain the glycine content of the biological sample.

Initial velocity method: and (3) treating a general biological sample, taking supernatant, adding 25 mu l of biological sample into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, slightly and uniformly mixing, reacting for 2-10 minutes at 30-40 ℃, and taking a blank control as ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader.

And calculating the glycine 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 glycine standard curve prepared by an initial speed 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 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, 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 Oxidase Glycine Oxidase and Glyoxylate Reductase Glycyxylate Reductase produced in the step (2);

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 11000-12000, 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, concentrating the protein at the temperature of 4-16 ℃ and at the rpm of 5000-6000, 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, 200 ml 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 Glycine Oxidase Glycine oxidases in the method for detecting Glycine by using the double-enzyme coupling method adopts the following coding gene sequences: firstly, the homology similarity of the gene sequence and the coding gene sequence of Glycine Oxidase Glycine Oxidase is more than 50%, and the coding protein has the gene sequence of Glycine Oxidase activity; second, amino acid sequence identity to glycine oxidase Glycineoxidase protein is greater than 40%, and the encoded protein has glycine oxidase activity.

Preferably, the glyoxylate reductase glyxylateReductase in the method for detecting glycine by the double enzyme coupling method adopts the following coding gene sequences: firstly, the homology similarity of the gene sequence and the coding gene sequence of Glyoxylate Reductase glyosylate Reductase is more than 50 percent, and the coding protein has the gene sequence of Glyoxylate Reductase activity; second, the amino acid sequence identity to the Glyoxylate Reductase glyoxysylate Reductase protein is greater than 40% and the encoded protein has Glyoxylate Reductase activity.

The invention also comprises the application of the method for detecting the glycine content by the enzyme method, which is used for developing a glycine detection kit. For example, according to the principle of the present invention for measuring glycine content by the double enzyme coupling method, GO and GR prepared by the procedure of the present invention, along with the components of flavin adenine dinucleotide disodium aqueous solution, NADH, buffer solution (100 mM TEA-HCl solution (pH 9.0), 100. mu.M flavin adenine dinucleotide disodium aqueous solution, 5 mM NADH solution), etc. are placed in a kit, and instructions for use are made according to the procedure of the present invention, whereby a kit can be easily developed.

As shown in fig. 1, the principle schematic diagram of the method for detecting Glycine content by enzyme method, Glycine is converted into glyoxylic acid and hydrogen peroxide by the action of Glycine Oxidase (GO); glyoxylate reductase (glyosylate reductase, GR) catalyzes the reduction of glyoxylate to glycolate with the oxidation of NADH to NAD⁺So that the absorbance of the system at 340nm is reduced; according to the detection method, except that flavin adenine dinucleotide disodium and NADH are purchased commercially, GO and GR need to be prepared, coding genes of GO and GR are determined by combining sequence comparison analysis means according to literature reports, target genes are obtained by whole-gene synthesis, an expression vector is connected, and the target genes are transferred into escherichia coli or other expression hosts special for protein expression, so that a large amount of exogenous expression, separation and purification of target proteins are realized;

secondly, constructing a reaction system containing GO, GR, flavin adenine dinucleotide disodium, glycine 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 glycine 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 glycine content by using the detection system; it should be noted that glycine has relevance to diabetes, brain diseases and other diseases, 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 Glycine content by the enzyme method overcomes the defect that the traditional Glycine Oxidase-horseradish peroxidase method is not suitable for blood environment, creatively oxidizes the Glycine to form glyoxylic acid by a double-enzyme coupling method of Glycine Oxidase Glycine Oxidase and glyoxylic acid Reductase Glycyxylate Reductase, and rapidly and correctly represents the Glycine content in the system by reducing the NADH content through the reduction reaction of the glyoxylic acid. The invention has the advantages of high sensitivity, rapid and stable detection, good repeatability, simple operation, low cost and wide application range.

The method for detecting the glycine content 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 glycine content by an enzymatic method;

FIG. 2 is a standard curve obtained by the endpoint method in example 3 at a glycine concentration of 0 to 100. mu.M.

FIG. 3 is a standard curve obtained by the initial velocity method in example 3 at a glycine concentration of 0 to 100. mu.M.

Detailed Description

The invention is further elucidated with reference to the drawings and the detailed description.

A method for detecting glycine content by an enzymatic method comprises the following steps:

exogenous expression and separation and purification of protein:

(1) combining a sequence comparison analysis means, selecting a gene coding sequence of Glycine Oxidase Glycine Oxidase and a gene coding sequence of Glyoxylate Reductase Glycosylate Reductase, and then obtaining a target gene of Glycine Oxidase Glycine Oxidase and a target gene of Glyoxylate Reductase Glycosylate Reductase by a method of synthesizing the target gene by whole genes;

(2) respectively connecting the Glycine Oxidase Glycine Oxidase target gene and Glyoxylate Reductase Glycyxylate Reductase target gene obtained in the step (1) into a pET28a expression vector by molecular cloning, respectively transferring pET28a-GO and pET28a-GR plasmids with correct sequencing after cloning into an escherichia coli Rosetta (DE3) strain, culturing the escherichia coli Rosetta strain in a test tube filled with Luria-Bertani (LB) culture medium at 37 ℃ for overnight, then transferring the Escherichia coli Rosetta strain into a shake flask filled with LB culture medium, and culturing the Escherichia coli Rosetta strain and the Escherichia coli Glyoxylate Reductase Glyoxylate Reductase target gene at 37 ℃ and 220 rpm until OD is OD₆₀₀Adding IPTG with the final concentration of 1mM when the concentration is about 0.6, and carrying out induced expression for 12 hours at the temperature of 16 ℃ to respectively obtain Glycine Oxidase target protein and Glyoxylate Reductase glycoxylate Reductase 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 a buffer solution A, and suspending the thalli 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, 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 Oxidase target protein and Glyoxylate Reductase target protein obtained in the step (2);

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 11000-12000, 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, concentrating the protein at the temperature of 4-16 ℃ and at the rpm of 5000-6000, 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, 200 ml 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 glycine by a double-enzyme coupling method:

(1) preparing a reaction mixed solution:

preparing 100 mM TEA-HCl solution (pH 9.0), 100 mu M flavin adenine dinucleotide disodium water solution and 5 mM ADH solution for later use; preparing 500 mu l of double-enzyme mixed solution containing Glycine Oxidase Glycine Oxidase and glyoxylate reductase Glycosylate Reductase, wherein the mixed solution contains 416 mu l of purified GO target protein with the concentration of 8.58mg/ml and 84 mu l of purified GR target protein with the concentration of 8.49mg/ml for later use;

preparing a reaction mixed solution according to the solution, wherein the proportion of each 175 mu l of the reaction mixed solution comprises the following components: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; 10-15 mul of double-enzyme mixed solution; make up to 175. mu.l of ultrapure water.

(2) The end-point method is used for preparing a glycine concentration standard curve:

preparing a series of glycine standard solutions with the concentration of 0-1.2 mM, respectively taking 25 mu l of glycine standard solution with each concentration, respectively dripping the glycine standard solutions into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, gently mixing the glycine standard solutions, reacting at the temperature of 30-40 ℃ for 20-40 minutes, and finishing the reaction. The reaction mixture of the blank control comprises the following components in proportion: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. mu.l. The proportional composition of the reaction mixture of the original absorbance reading includes: TEA-HCl solution, 100. mu.l; 10-30 mul of flavin adenine dinucleotide disodium water solution; 8-10 mul of NADH solution; 10-15 mul of double-enzyme mixed solution; make up ultrapure water to 200. 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. Drawing a glycine concentration standard curve by taking the absorbance change value as a vertical coordinate and the concentration of the glycine standard solution as a horizontal coordinate;

(3) initial velocity method for preparing glycine concentration standard curve

Preparing a series of glycine standard solutions with the concentration of 0-1.2 mM, respectively taking 25 mu l of glycine standard solution with each concentration, respectively dripping the glycine standard solutions into a 96-hole enzyme label plate filled with 175 mu l of reaction mixed solution, gently mixing the glycine standard solutions, and reacting for 2-10 minutes at the temperature of 30-40 ℃. The blank control was ultrapure water. The change in absorbance at 340nm was monitored using a microplate reader. And drawing a glycine concentration standard curve by taking the absorbance change value at 340nm within 2-10 minutes as a vertical coordinate and taking the concentration of the glycine 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;

the English name of the Flavin adenine dinucleotide disodium is Flavin adenine dinucleotide sodium salt, CAS number 84366-81-4 (anhydrous);

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.