CN114480571A - Fructose detection method based on fructokinase reaction system - Google Patents

Fructose detection method based on fructokinase reaction system Download PDF

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CN114480571A
CN114480571A CN202210395204.4A CN202210395204A CN114480571A CN 114480571 A CN114480571 A CN 114480571A CN 202210395204 A CN202210395204 A CN 202210395204A CN 114480571 A CN114480571 A CN 114480571A
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fructose
detection
fructokinase
concentration
reaction system
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李长红
朱秋莎
周雪
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Nanjing Shengde Ruier Pharmaceutical Technology Co ltd
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Nanjing Shengde Ruier Pharmaceutical Technology Co ltd
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    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01004—Fructokinase (2.7.1.4)
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Abstract

The invention discloses a fructose detection method based on a fructokinase reaction system, which belongs to the technical field of detection, is based on the activity of fructokinase FK and adopts the principle of three-step enzyme kinetics method determination, and specifically comprises the following steps of step one, plasmid construction of fructokinase genes and expression and purification of fructokinase protein; step two, enzymatic detection: constructing a fructose detection method on the basis of FK enzyme kinetic detection; and step three, detecting the fructose in the standard product by taking substitutes of blood, urine and beverage as examples, further detecting the fructose content of the sample, realizing high-specificity detection of the fructose, and having a wide detection range, thereby having good application prospect.

Description

Fructose detection method based on fructokinase reaction system
Technical Field
The invention relates to the technical field of detection, in particular to a fructose detection method based on a fructokinase reaction system.
Background
Fructose is one of the most common ketohexoses found widely in nature. Fructose contains 6 carbon atoms, is also a monosaccharide, is an isomer of glucose, and is a component of sucrose.
After the human body takes fructose, the fructose is absorbed by intestinal tracts and directly enters cells to be metabolized without insulin, and the fructose is one of important factors causing fat deposition in human organs. When too much fructose is present in the diet, it can be potentially dangerous to the liver, arteries, and heart. There are also diabetes patients who have elevated levels of fructose in the blood circulation, congenital fructodiabetes caused by loss-of-function mutations in fructokinase, and patients who have elevated levels of fructose in the blood after eating a diet rich in fructose and excreted in urine.
However, the existing fructose detection means has low specificity and limited detection range, so a fructose detection method based on a fructokinase reaction system is provided.
Disclosure of Invention
1. Technical problem to be solved
In view of the problems in the prior art, the invention aims to provide a fructose detection method based on a fructokinase reaction system, which is based on the principle that the FK activity of fructokinase is measured by a three-step enzyme kinetics method, and comprises the following steps: phosphorylation of fructose to fructose-6-phosphate by FK with ATP as a cofactor; the second step is that: ADP formed in the reaction process is used by pyruvate kinase to convert phosphoenolpyruvate into pyruvate; the third step: the lactate dehydrogenase uses NADH as a cofactor to convert pyruvic acid into lactic acid, and the FK activity is measured by tracking the decrease of absorbance of NADH at 340nm, so that the fructose detection specificity is high, and the detection range is wide.
2. Technical scheme
In order to solve the above problems, the present invention adopts the following technical solutions.
The invention relates to a fructose detection method based on a fructokinase reaction system, which is based on the activity of fructokinase FK and adopts the principle of three-step enzyme kinetics method determination, and the method specifically comprises the following steps of, step one, plasmid construction of fructokinase gene and expression and purification of fructokinase protein; step two, enzymatic detection: constructing a fructose detection method on the basis of FK enzyme kinetic detection; and step three, detecting the fructose in the standard substance by taking substitutes of blood, urine and beverage as examples, and further detecting the fructose content in the sample to realize high-specificity detection of the fructose.
Further, in the detection method:
step one, plasmid construction of fructokinase gene and expression and purification of fructokinase protein comprise:
s1, FK protein prokaryotic expression:
s11, construction of a prokaryotic expression system: synthesizing a target gene according to a standard procedure, using an escherichia coli expression vector plasmid pET-28a, inserting an FK protein gene into an enzyme cutting site Xho I/Nco I to construct a recombinant expression vector pET-28a-FK, screening a kanamycin (kan) resistance gene, and purifying HIS tag protein;
s12, expression and purification of prokaryotic recombinant protein;
step two, enzymatic detection: the fructose detection method constructed on the basis of FK enzyme kinetic detection comprises the following steps:
s2, establishing an FK enzyme kinetic detection method:
chemical to be evaluated: d- (-) -fructose, D- (+) -glucose, sucrose;
s22, detection equipment: CLARIOstar multifunctional enzyme labeling instrument;
s23, setting program: the reaction mixture included 100 mM Tris-HCl (pH 7.5), 1 mM Fructose, 1 mM ATP, 100 mM KCl, 1.5 mM MgCl2, 1 mM PEP, 0.5 mM NADH, 0.2 μ l PK/LDH, and 10 μ l of purified recombinase, with a final reaction capacity of 100 μ l;
s24, FK activity assay: firstly, selecting two FKs with different concentrations to detect fructose, setting 12 concentration gradients in a concentration range of 0-10 mM in a system, starting to dilute by multiple from the highest concentration of 250 mM (the final concentration in the reaction system is 10 mM) and setting the fructose concentration to be 0mM as a blank control, starting to continuously measure the reaction by adding substrates with different concentrations in a CLARIOstar multifunctional enzyme-labeling instrument, measuring the activity by tracking the change of absorbance at 340nm, which corresponds to the conversion of NADH to NAD +, performing the measurement for 30 minutes at 25 ℃, inputting corresponding values of detection results of the fructose with different concentrations into Graphpad Prism software for analysis, performing the analysis by using regression line fitting of a linear equation, and determining the average value +/-standard error (n = 4);
step three, taking substitutes of blood, urine and beverage as examples to detect the fructose of the standard substance, and further detecting the fructose content of the sample:
s3, an experimental pretreatment method and an enzymatic detection method by using blood, urine and beverage substitutes as fructose solvents.
Further, the step S12 includes the following steps:
firstly, transforming into an expression bacterium:
adding pET-28a-FK plasmid into BL21 (DE 3) competence for mixing, and then transforming according to the conventional process;
secondly, inducing expression in batches:
and selecting a single colony for amplification and batch expression, wherein the antibiotic is kanamycin, and the single colony is induced by IPTG.
Further, Purification was carried out by a standard method of His-tag Purification Resin, and protein purity was checked by SDS-PAGE electrophoresis.
Further, the step S3 of detecting the content of fructose in serum of blood includes the following steps:
firstly, sample pretreatment: for detecting the fructose content in blood, FBS (fetal bovine serum) and plasma analogue (r-SBFA) are used as solvents to dissolve fructose with different concentrations, 45 mu l of the plasma analogue (r-SBFA) is added into a 1.5 ml EP tube, 5 mu l of fructose with different concentrations and plasma analogue (r-SBFA) as a solvent is added, 150 mu l of methanol solution precooled at 4 ℃ is added into each tube, vortex and mix evenly for 5min, 13000g (4 ℃, 15 min) is centrifuged after standing for 60 min at-20 ℃, supernatant is taken and added into a FK reaction system, and the fructose concentration is detected;
secondly, establishing a standard curve: in the supernatant of the pretreated sample, the highest concentration of fructose is set to be 100 mM, the final concentration in the diluted reaction system is 5 mM, 12 concentration gradients are set, dilution is carried out by multiple times from the highest concentration, and the fructose concentration is set to be 0mM as blank control;
thirdly, sample detection: three samples (n = 3) at different concentrations were chosen to test the accuracy of this protocol.
Further, the step S3 of detecting the content of fructose in urine includes the following steps:
firstly, preparing artificial urine (MP-AU): the relevant reagents were added to 100ml of ultrapure water in the order provided in the table below;
TABLE 1
Reagent Molarity (mM) Quantity (g/100ml)
Na2SO4 11.965 0.17
C5H4N4O3 1.487 0.025
Na3C6H5O7·2H2O 2.45 0.072
C4H7N3O 7.791 0.0881
CH4N2O 249.75 1.5
KCl 30.953 0.2308
NaCl 30.053 0.1756
CaCl2·2H2O 1.663 0.0244
NH4Cl 23.667 0.1266
K2C2O4 0.19 0.0035
MgSO4 4.389 0.0449
NaH2PO4·2H2O 18.667 0.224
Na2HPO4·2H2O 4.667 0.1653
Secondly, establishing a standard curve: weighing a proper amount of fructose to be dissolved in artificial urine (MP-AU), setting the highest concentration of the fructose to be 100 mM, setting the final concentration in a diluted reaction system to be 5 mM, setting 12 concentration gradients, starting to dilute by multiple times from the highest concentration and setting the concentration of the fructose to be 0mM as blank control;
thirdly, sample detection: three samples (n = 3) at different concentrations were chosen to test the accuracy of this protocol.
Further, the detection of the content of fructose in the beverage in the step S3 includes the following steps:
firstly, establishing a standard curve: weighing a proper amount of fructose, dissolving the fructose in an Ultra-pure water, setting the highest concentration of the fructose to be 100 mM, setting the final concentration of the fructose in a diluted reaction system to be 5 mM, setting 12 concentration gradients, carrying out multiple dilution from the highest concentration and setting the fructose concentration to be 0mM as blank reference;
secondly, sample detection: three samples with different concentrations were selected to test the accuracy of this protocol.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
the present scheme is based on the principle that Fructokinase (FK) activity is determined by a three-step enzyme kinetics method, the first step: phosphorylation of fructose to fructose-6-phosphate by FK with ATP as a cofactor; the second step is that: the ADP formed during the reaction is used by pyruvate kinase, which converts phosphoenolpyruvate to pyruvate; the third step: lactate dehydrogenase converts pyruvate into lactate using NADH as a cofactor, measures FK activity by tracking the decrease in absorbance of NADH at 340nm, and is purified by the following steps, step one, plasmid construction of fructokinase gene and expression of fructokinase protein; step two, enzymatic detection: constructing a fructose detection method based on FK enzyme kinetic detection, and preparing a standard curve and a calculation method for calculating the fructose concentration of a sample based on standard product detection mapping; and step three, detecting the fructose in the standard product by taking substitutes of blood, urine and beverage as examples, further detecting the fructose content of the sample, realizing high-specificity detection of the fructose, and having a wide detection range, thereby having good application prospect.
Drawings
FIG. 1 is a schematic diagram of an enzyme coupling reaction for determining Fructokinase (FK) activity according to the present invention;
FIG. 2 is a schematic representation of a map of the pET-28a-FK plasmid of the present invention;
FIG. 3 is a schematic representation of the results of SDS-PAGE gel electrophoresis of different batches of protein eluate of the present invention;
FIG. 4 is a standard curve of the protein of the present invention;
FIG. 5 is a line graph showing consumption of NADH by FK reaction in the presence of fructose according to the present invention;
FIG. 6 is a line graph showing the detection of the specificity of FKs of the present invention;
FIG. 7 is a standard curve of FK reaction with FBS as solvent for fructose of the present invention;
FIG. 8 is a graph showing the FK reaction standard curve of the fructose in the solvent of r-SBFA;
FIG. 9 is a standard curve of FK reaction with MP-AU (5 μ l) as solvent for fructose of the present invention;
FIG. 10 is a FK reaction standard curve of fructose in MP-AU (10. mu.l) as solvent;
FIG. 11 is a standard curve of FK reaction of fructose in the present invention using Ultra-pure water as a solvent.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention; it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by those skilled in the art without any inventive work are within the scope of the present invention.
As shown in fig. 1, the present protocol is based on the principle that Fructokinase (FK) activity is determined by three-step enzyme kinetics, the first step: phosphorylation of fructose to fructose-6-phosphate by FK with ATP as a cofactor; the second step: the ADP formed during the reaction is used by pyruvate kinase, which converts phosphoenolpyruvate to pyruvate; the third step: lactate dehydrogenase converts pyruvate into lactate using NADH as a cofactor, measures FK activity by tracking the decrease in absorbance of NADH at 340nm, and is purified by the following steps, step one, plasmid construction of fructokinase gene and expression of fructokinase protein; step two, enzymatic detection: constructing a fructose detection method based on FK enzyme kinetic detection, and preparing a standard curve and a calculation method for calculating the fructose concentration of a sample based on standard product detection mapping; and step three, detecting the fructose in the standard substance by taking substitutes of blood, urine and beverage as examples, and further detecting the fructose content in the sample to realize high-specificity detection of the fructose.
Example (b):
first, FK protein prokaryotic expression
1. Construction of prokaryotic expression System
The target gene was synthesized according to the standard procedure, and the recombinant expression vector pET-28a-FK was constructed by inserting FK protein gene into E.coli expression vector plasmid pET-28a at the restriction site Xho I/Nco I, selecting kanamycin resistance gene, and purifying HIS tag protein, as shown in FIG. 2.
2. Expression and purification of prokaryotic recombinant protein
2.1 expression of the protein
(1) Transformation into expression bacteria by conventional method
1. 2 μ L of pET-28a-FK plasmid was added to the 50 μ L BL21 (DE 3) competence on an ultraclean bench and mixed for 30 min in an ice bath.
2. Heat shock: placing the mixture in water bath, heating in 42 deg.C water bath for 90s, and taking out quickly.
3. After ice-bath for 3 min, 900 μ L of liquid LB medium without antibiotics was added to the mixture on an ultraclean bench and placed on a shaker at 37 ℃ and shaken at 220 rpm for 45 min.
The mixture was centrifuged at 4.2000 rpm for 2 min.
5. Discarding a large part of supernatant on an ultra-clean workbench, reserving about 50 mu L of culture solution to resuspend the thalli, sucking out and adding into a solid LB culture plate with Kan antibiotics (50 mu g/mL), uniformly coating the bacteria in a ball coating method, inverting and putting into an incubator at 37 ℃ for overnight culture.
(2) Inducible expression
1. In the clean bench, single colonies were picked from the plates and cultured overnight at 37 ℃ with 5 mL of liquid LB medium containing Kan antibiotics (50. mu.g/mL) at 220 rpm.
2. The culture medium is operated in a clean bench, the overnight cultured bacterial liquid is transferred to a conical flask containing 200 mL of liquid LB culture medium, Kan antibiotics are added, and the culture is carried out at 37 ℃ and the rotating speed of 220 r/min.
3. When the culture is carried out for about 3-4h, 1 mL of bacterial liquid is sucked from the culture to detect the OD600 value until the OD600 is between 0.6 and 0.8, an inducer IPTG (100 mM) is added into a conical flask according to the proportion of 1:1000 for induction expression, and the culture is carried out for about 12h at the temperature of 37 ℃ and the rotating speed of 220 r/min.
4. Collecting bacterial liquid: centrifuging at 4 ℃ and 12000 r/min for 20 min, discarding the supernatant, washing with PBS once, centrifuging at 44 ℃ and 12000 r/min for 20 min, and discarding the supernatant.
2.2 purification of the protein
The recombinant protein FK carries the HIS tag and the protein is purified using the HIS tag protein purification kit.
a. And (3) centrifugally collecting the bacterial sediment, adding a lysis solution according to the proportion that 4 ml (2-5 ml) of non-denatured lysis solution is added into each gram of bacterial sediment in a wet weight manner, and fully suspending the thalli. If necessary, an appropriate amount of protease inhibitor cocktail, such as the P1030/P1031 protease inhibitor cocktail from Byunyan (His-Tag protein for purification, 100X), can be added to the lysate prior to lysing the bacteria.
Note: the subsequent reagent amounts were 1 gram of bacteria and 4 ml of lysate.
b. Adding lysozyme to final concentration of 1 mg/ml, mixing, and standing in ice water bath or ice for 30 min.
Note: lysozyme can be prepared into a mother solution of 100 mg/ml by using a lysis solution and added before use. The lysozyme is prepared into mother liquor, and can be properly subpackaged and stored at-20 ℃.
c. Bacteria were lysed ultrasonically on ice. The ultrasonic power is 200-300W, each time of ultrasonic treatment is 10s, each time interval is 10s, and the ultrasonic treatment is carried out for 6 times.
Note: the specific ultrasonic treatment mode needs to be searched and optimized according to the ultrasonic instrument with a specific model.
(optionally) if the lysate is very viscous after the ultrasonic treatment, RNase A to 10 mug/ml and DNase I to 5 mug/ml may be added and left on ice for 10-15 min. Alternatively, aspiration may be repeated several times using a syringe equipped with a thin needle as appropriate to shear viscous genomic DNA or the like.
e.4 deg.C, centrifuging at 10000g for 20-30 min, collecting bacterial lysate supernatant, and placing on ice water bath or ice. 20 mul of supernatant can be taken for subsequent detection.
Note: the supernatant must be kept clear, i.e. free of any insoluble material, for further purification, and if it is contaminated with insoluble impurities it will seriously affect the purity of the protein obtained by subsequent purification.
f. 1 ml of the mixture was used to purify the expressed protein by using 50% of a Beyogold His-tag Purification Resin (denaturation resistant form).
Centrifuging at 4 deg.C (1000g × 10s) to remove stock solution, adding 0.5 ml of non-denaturing lysis solution into the gel, mixing to balance the gel, centrifuging at 4 deg.C (1000g × 10s) to remove liquid, repeating the balancing for 1-2 times, and removing liquid. About 4 ml of bacterial lysate supernatant was added thereto and shaken slowly on a side or horizontal shaker at 4 ℃ for 60 min.
Note: the Beyogold His-tag Purification Resin can be used directly without being balanced, but the yield of the protein may be reduced by 5-20%.
g. A mixture of the lysate and the Beyogold. DELTA.His-tag Purification Resin (denaturation resistant formulation) was loaded into an affinity chromatography column empty tube provided in the kit.
Note: or 1 ml of uniformly mixed 50% Beyogold His-tag Purification Resin (denaturation resistant dosage form) can be taken firstly for column packing, then 0.5 ml of non-denaturing lysis solution is used for balancing for 2-3 times, about 4 ml of bacterial lysis solution supernatant is added, and subsequently the flow-through solution can be collected and repeatedly loaded on the column for 3-5 times to fully combine the target protein. The mode of mixing and then loading the mixture into the column is relatively troublesome to operate, but is more favorable for the full combination of the recombinant protein with the His label and the nickel column, and particularly, the combination efficiency of the recombinant protein with the His label and the nickel column is higher when the His label is partially shielded by the protein or the concentration of the recombinant protein with the His label is low.
h. And opening a cover at the bottom of the purification column, allowing liquid in the column to flow out under the action of gravity, and collecting about 20 mu l of flow-through liquid for subsequent analysis.
i. And (3) washing the column for 5 times, adding 0.5-1 ml of non-denaturing washing liquid each time, collecting about 20 mu l of washing liquid penetrating the column each time for subsequent analysis and detection, and simply and quickly detecting the protein content in the washing liquid and the eluent each time by using a Bradford method (P0006) in the processes of column washing and next elution, thereby considering the increase or reduction of the times of washing and elution.
Note: if the subsequent protein obtained has not high enough purity, the column washing times can be increased for 2-3 times.
j. Eluting the target protein for 6-10 times, and eluting with 0.5 ml of non-denatured eluent each time. And respectively collecting the eluates in each time into different centrifuge tubes, and collecting the obtained eluates, namely the purified His tag protein samples.
3. SDS-PAGE gel electrophoresis
3.1 filling and sampling
The glass plates are placed into the clamp for clamping after being aligned, then the glass plates are vertically clamped on the frame for glue pouring, and the two pieces of glass are aligned during operation so as to avoid glue leakage.
(1) Glue preparation
Preparation of separation gel and concentrated gel: according to the sequence and proportion of the solutions in the following table, 12% of separation gel and 5% of concentrated gel are prepared.
TABLE 2 preparation method of separation gel
Reagent 12% separation gel (10 mL)
30％Acr-Bis（mL） 4
1.5M Tirl-HCI pH8.8（mL） 2.5
ddH2O（mL） 3.3
10%SDS（μL） 100
10%APS（μL） 100
TEMED（μL） 6
TABLE 3 preparation of concentrated gums
Reagent 5% separation gel (10 mL)
30％Acr-Bis（mL） 0.66
1.5M Tirl-HCI pH6.8（mL） 0.5
ddH2O（mL） 2.75
10%SDS（μL） 40
10%APS（μL） 40
TEMED（μL） 4
(2) Step of compounding glue
And (3) preparing 12% separation glue, adding TEMED, immediately shaking up to fill the glue, sucking a proper amount of glue by using a 3 ml suction pipe or a 10 ml gun to discharge along the glass when the glue is filled, and adding a layer of water when the glue surface rises to the height of the middle line of the green belt, so that the gelation is quicker after liquid sealing. (the glue can be poured quickly, the glue surface is slowed down when reaching the required height, the glue must flow down along the glass plate during operation, so that no air bubbles exist in the glue, the water is added for liquid sealing, otherwise, the glue is punched and deformed.)
When a refracted ray exists between the water and the glue, the glue is solidified, and the glue can be poured out after 3 min to fully solidify the glue, and the water is sucked to be dry by using water absorption paper.
Preparing 5% of concentrated glue according to the method, adding TEMED, immediately shaking up to fill the glue, filling the residual space with the concentrated glue, then inserting a comb into the concentrated glue, and making the glue flow down along a glass plate during glue filling so as to avoid bubbles in the glue. The comb is required to be kept horizontal when inserted, the volume is reduced due to the fact that the volume can shrink when the glue is solidified, and therefore the sample loading volume of the sample adding hole is reduced, the glue is frequently supplemented on two sides in the process of solidification of the concentrated glue, and after the concentrated glue is solidified, two hands respectively hold two sides of the comb to vertically and upwards gently pull out the comb.
(3) Sample loading
The concentrated gel was washed with water and placed in an electrophoresis tank. The sample was removed to a 0.5 ml centrifuge tube and 5 x SDS loading buffer was added to a final concentration of 1 x, before loading the sample was boiled in boiling water for 5min to denature the protein.
Sufficient running solution is added to begin the preparation of the sample. And sucking the sample by using a microsyringe in an adherent manner, sucking the sample out without sucking air bubbles, and inserting a needle of the microsyringe into the sample adding hole to slowly add the sample.
3.2 electrophoresis
After sample adding, covering the upper cover, connecting the electrophoresis apparatus, opening a switch of the electrophoresis apparatus, and controlling the voltage to be 100-200V for about 15-20 min before the sample enters the gel; after the bromophenol blue indicator in the sample reaches the separation gel, the voltage is raised to 200V, the electrophoresis process keeps stable, electrophoresis is stopped when the bromophenol blue indicator migrates to a position 1-2 cm away from the front edge, the electrophoresis is stopped for about 0.5-1 hour, if the room temperature is high, the circulating water of the electrophoresis tank is opened, and the electrophoresis temperature is reduced.
3.3 dyeing and decolorizing
After the electrophoresis is finished, the power supply is turned off, the glass plates are taken out, the gaps of the lower corners of the two long and short glass plates are slightly pried by a knife, the rubber surface is separated from one glass plate, then the rubber sheet is slightly supported, the copper wire is inserted into the center of the indicator zone to be used as a mark, the indicator zone is placed into a large culture dish for dyeing, 0.25% of Coomassie brilliant blue dye solution is used, the dyeing is carried out for 2-4 h, and the night can be kept when necessary.
Discarding the staining solution, rinsing the glue surface with distilled water for several times, then adding the destaining solution, performing diffusion destaining, and frequently changing the destaining solution until the protein band is clear.
Referring to FIG. 3, the FK protein was approximately 35 Kd and the His protein was approximately 0.6 Kd.
4. Protein concentration determination
4.1 preparation of protein Standard Curve
1. Taking out the frozen 5 mg/ml protein standard (Biyunyan, P0007) from-20 ℃, completely melting at room temperature and uniformly mixing for later use;
2. protein standards of 0, 0.125, 0.25, 0.5, 0.75, 1, 1.5 mg/ml were prepared according to the following table, both protein standards and samples were dissolved in 0.9% NaCl, and carefully mixed well at each dilution to obtain a protein standard curve (see fig. 4).
TABLE 4 protein Standard Curve construction
Numbering Volume of dilution liquid Volume of standard Final concentration
1 70μl 5 mg/ml BSA 30μl 1.5 mg/ml
2 30μl 60 μ l from 1 tube 1 mg/ml
3 20μl 60 μ l from 2 tubes 0.75 mg/ml
4 30μl 60 μ l from 3 tubes 0.5 mg/ml
5 60μl 60 μ l from 4 tubes 0.25 mg/ml
6 60μl 60 μ l from 5 tubes 0.125 mg/ml
7 60μl 0μl 0 mg/ml
4.2. Protein concentration determination
1. Adding 5 mul of protein standards with different concentrations into a protein standard hole of a 96-well plate;
2. taking 5 mul of sample to a sample well of a 96-well plate;
3. add 250 u l G250 staining solution into each well;
4. measuring the absorbance of the A595 wave length by using an enzyme labeling instrument, and finishing the measurement within 2 hours;
5. the protein concentration in the sample was calculated from the standard curve and the sample volume used.
TABLE 5 measurement of protein concentration in eluate
Batch of eluent Concentration (mg/ml)
E1 1.051
E2 2.500
E3 3.000
E4 0.285
E5 0.180
Second, establishing FK enzyme dynamic detection method
1. Materials and reagents
96-well plate
Purified recombinant His-tagged FK
PK/LDH（PK/LDH enzymes from rabbit muscle）
100 mM PEP (Phospho(enol)pyruvic acid tri(cyclohexylammonium) salt)
1 M KCl；
100 mM ATP；
1 M Tris-HCl（pH 7.5）；
10 mM β-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH)；
100 mM MgCl2；
1 M KCl；
1. Chemical to be evaluated:
a. (iv) D- (-) -fructose;
b. d- (+) -glucose;
c. sucrose.
2. Device
CLARIOstar multifunctional enzyme-labeling instrument
3. Procedure for measuring the movement of a moving object
1. The reaction mixture included 100 mM Tris-HCl (pH 7.5), 1 mM fructose, 1 mM ATP (now ready to use), 100 mM KCl, 1.5 mM MgCl 21 mM PEP, 0.5 mM NADH (now ready for use), 0.2 μ l PK/LDH (stored at-20 ℃ C. before use), and 10 μ l of purified recombinase, with a final reaction capacity of 100 μ l.
TABLE 6 composition of reaction mixture for determining FK Activity
Stock solutions Added in reaction mix (μl)
Tris-HCl (pH 7.5) (1 M) 10
Fructose (20 mM) 4
ATP (100 mM) 1
MgCl2(100 mM) 1.5
KCl (1 M) 10
PEP (100 mM) 1
NADH (10 mM) 2
PK/LDH (600-1,500 units/ml) 0.2
FK 10
Ultra-pure water 60.3
FK Activity detection assay
As shown in FIG. 1, to calculate the enzyme kinetic parameters of the selected substrates, separate enzyme reactions were performed in the presence of at least five different concentrations of substrate and a fixed concentration of ATP. Wherein, fructose is a specific substrate of FK, two different concentrations of FK are firstly selected for detecting fructose, the concentration range in the system is 0-10 mM, 12 concentration gradients are set, dilution is carried out by multiple times from the highest concentration of 250 mM (10 mM in the system) and the fructose concentration is set as a blank control, and the method is characterized in that the concentration of the FK is controlled by the concentration of 0 mM.
The reaction was continuously measured in a CLARIOstar multifunctional microplate reader by adding different concentrations of substrate and activity was measured by following the change in absorbance at 340nm, which corresponds to the conversion of NADH to NAD +, which was carried out at 25 ℃ for 30 minutes, resulting in a reaction curve as shown in fig. 5.
The corresponding values for the different concentrations of fructose assay were input to Graphpad Prism software for analysis, and analyzed using regression line fitting of the line equation to determine the mean ± standard error (n = 4) (see fig. 5).
To determine substrate specificity, FK activity was evaluated in the presence of each of the substrates listed in the "materials and reagents" section, and it was found from the experimental results shown in fig. 6 that purified FK is specific only to fructose.
Third, other detection field expansion
In order to apply the detection method of the invention to other sample detection services, the detection items with higher attention in the medical and health fields at present, such as the content of fructose in blood, urine and beverages, are selected.
1. Detection of fructose content in serum
In order to detect the content of fructose in blood, FBS (fetal bovine serum) and plasma analogue (r-SBFA) are used as solvents to dissolve fructose with different concentrations, and after sample pretreatment, a detection standard curve is prepared by adding the solution into a reaction system of an established FK detection method.
TABLE 7 plasma analogs (r-SBFA) formulation method
Reagent Quantity (g/1L) Quantity (g/100ml)
NaCl 5.403 0.540
NaHCO3 0.740 0.074
Na2CO3 2.046 0.205
KCl 0.225 0.023
KH2PO4 0.138 0.014
MgCl2 0.143 0.0143
HEPES 11.928 1.193
CaCl2·2H 20 0.388 0.039
NaSO4 0.072 0.007
BSA 40.000 4.000
(1) Sample pretreatment
Mu.l of the plasma analogue (r-SBFA) is added into a 1.5 ml EP tube, 5. mu.l of fructose with different concentrations and taking the plasma analogue (r-SBFA) as a solvent is added into each tube, 150. mu.l of methanol solution precooled at 4 ℃ is added into each tube, vortex and mix uniformly for 5min, 13000g (4 ℃, 15 min) are centrifuged after standing for 60 min at-20 ℃, supernatant is taken and added into an FK reaction system, and the fructose concentration is detected.
(2) Standard curve creation
In the supernatant of the pretreated sample, the highest concentration of fructose is set to be 100 mM, the adding amount of each hole is changed to be 10. mu.l, the corresponding adding amount of Ultra-pure water is changed to be 54.3. mu.l, the final concentration after the fructose is added into the reaction system is 5 mM, 12 concentration gradients are set, dilution is carried out by multiple proportions from the highest concentration, and the fructose concentration is set to be 0mM as a blank control.
(3) Sample detection
Three samples (n = 3) with different concentrations were selected to test the accuracy of the experimental scheme, the coefficient of variation (coefficient of variation) was small, and the experimental results were reliable (see fig. 7 and 8).
C.v = sd/mean ×100%；
C.V = coefficient of variation;
SD = standard deviation;
mean = Mean.
TABLE 8 statistical analysis of sample test results
Sample (I) Mean SD CV
Sample
1 274.2553 5.8758 2.1424
Sample 2 269.6460 10.1771 3.7743
Sample 3 315.6157 15.7200 4.9807
2. Detection of fructose content in urine
In order to detect the content of fructose in urine, artificial urine (MP-AU) is used as a solvent to dissolve fructose with different concentrations, and after sample treatment, the sample is added into a reaction system of an established FK detection method to prepare a detection standard curve.
TABLE 9 preparation method of artificial urine (MP-AU)
Reagent Molarity (mM) Quantity (g/100ml)
Na2SO4 11.965 0.17
C5H4N4O3 1.487 0.025
Na3C6H5O7·2H2O 2.45 0.072
C4H7N3O 7.791 0.0881
CH4N2O 249.75 1.5
KCl 30.953 0.2308
NaCl 30.053 0.1756
CaCl2·2H2O 1.663 0.0244
NH4Cl 23.667 0.1266
K2C2O4 0.19 0.0035
MgSO4 4.389 0.0449
NaH2PO4·2H2O 18.667 0.224
Na2HPO4·2H2O 4.667 0.1653
(1) The key point of preparing artificial urine (MP-AU)
The relevant reagents were added to 100ml of ultrapure water in the order provided in the above table, and the mixing was performed with the magnetic stirrer set to a rotation speed of 500 rpm, during which the temperature of the solution was maintained at 37.5 ℃ using the heating function of the stirrer.
The pH value of the MP-AU solution is stabilized to about 6.00 +/-0.08 after 24h at 37 ℃, and the preparation is completed 24h before the experiment because the experiment has strict requirements on the pH value.
(1) Standard curve creation
Weighing a proper amount of fructose to be dissolved in artificial urine (MP-AU), setting the highest concentration of the fructose to be 100 and 200 mM, changing the adding amount of each hole to be 5 mul/10 mul in order to reduce the influence of the pH of the artificial urine (MP-AU) on the reaction, correspondingly changing the Ultra-pure water in the system to be 59.3 mul/54.3 mul, setting the final concentration to be 5 mM after being added into the reaction system, setting 12 concentration gradients, carrying out dilution by multiple times from the highest concentration and setting the fructose concentration to be 0mM as blank control.
(2) Sample detection
Three samples (n = 3) with different concentrations are selected to detect the accuracy of the experimental scheme, the coefficient of variation is small, and the experimental result is reliable, as shown in fig. 9-10.
C.v = sd/mean ×100%；
C.V = coefficient of variation (coefficient of variation);
SD = standard deviation;
mean = Mean.
TABLE 10 statistical analysis of sample test results
Sample (I) Mean SD CV
Sample No. 1 123.2997 4.3116 3.4969
Sample 2 174.0023 3.2593 1.8731
Sample 3 191.2873 4.3116 2.2540
3. Detection of fructose content in beverage
In order to detect the fructose content in daily beverages, Ultra-pure water is used as a solvent to dissolve fructose with different concentrations to prepare sugar water which is used as a beverage substitute, and after sample treatment, the sugar water is added into a reaction system of an established FK detection method to prepare a detection standard curve.
(1) Standard curve establishment
Weighing a proper amount of fructose, dissolving the fructose in an Ultra-pure water, setting the highest concentration of the fructose to be 100 mM, changing the adding amount of each hole to be 10 mu l, and correspondingly changing the Ultra-pure water in the system to be 54.3 mu l. After the addition to the reaction system, the final concentration was 5 mM, 12 concentration gradients were set, dilution was performed at double ratios starting from the highest concentration and the fructose concentration was set to 0mM as a blank.
(2) Sample detection
Three samples with different concentrations are selected to detect the accuracy of the experimental scheme, the variation coefficient is small, and the experimental result is reliable, as shown in fig. 11.
C.V = sd/mean ×100%；
C.V = coefficient of variation (coefficient of variation);
SD = standard deviation;
mean = Mean.
TABLE 11 statistical analysis of sample test results
Sample (I) Mean SD CV
Sample
1 244.1710 5.9984 2.4566
Sample 2 275.4077 1.6296 0.5917
Sample 3 323.8057 1.6296 0.5033
The scheme is based on the principle that the activity of Fructokinase (FK) is measured by a three-step enzyme kinetics method, and comprises the following steps of firstly, constructing plasmid of a fructokinase gene and expressing and purifying the fructokinase protein; step two, enzymatic detection: constructing a fructose detection method based on FK enzyme kinetic detection, and preparing a standard curve and a calculation method for calculating the fructose concentration of a sample based on standard product detection mapping; and step three, detecting the fructose in the standard product by taking substitutes of blood, urine and beverage as examples, further detecting the fructose content in the sample, realizing high-specificity detection of the fructose, and having a wide detection range.
The foregoing is only a preferred embodiment of the present invention; the scope of the invention is not limited thereto. Any person skilled in the art should be able to cover the technical scope of the present invention by equivalent or modified solutions and modifications within the technical scope of the present invention.

Claims (7)

1. A fructose detection method based on a fructokinase reaction system is characterized by comprising the following steps: the detection method is based on the activity of fructokinase FK and is based on the principle of three-step enzyme kinetic assay, and the method specifically comprises the following steps of firstly, constructing plasmid of fructokinase genes and expressing and purifying fructokinase protein; step two, enzymatic detection: constructing a fructose detection method on the basis of FK enzyme kinetic detection; and step three, detecting the fructose in the standard substance by taking substitutes of blood, urine and beverage as examples, and further detecting the fructose content in the sample to realize high-specificity detection of the fructose.
2. The fructose detection method based on the fructokinase reaction system as claimed in claim 1, which is characterized in that: the detection method comprises the following steps:
the first step, the plasmid construction of fructokinase gene and the expression and purification of fructokinase protein comprise:
s1, FK protein prokaryotic expression:
s11, construction of a prokaryotic expression system: synthesizing a target gene according to a standard procedure, using an escherichia coli expression vector plasmid pET-28a, inserting an FK protein gene into an enzyme cutting site Xho I/Nco I to construct a recombinant expression vector pET-28a-FK, screening a kanamycin (kan) resistance gene, and purifying HIS tag protein;
s12, expression and purification of prokaryotic recombinant protein;
step two, enzymatic detection: the fructose detection method constructed on the basis of FK enzyme kinetic detection comprises the following steps:
s2, establishing an FK enzyme kinetic detection method:
chemical to be evaluated: d- (-) -fructose, D- (+) -glucose, sucrose;
s22, detection equipment: CLARIOstar multifunctional enzyme labeling instrument;
s23, setting program: the reaction mixture included 100 mM Tris-HCl (pH 7.5), 1 mM Fructose, 1 mM ATP, 100 mM KCl, 1.5 mM MgCl2, 1 mM PEP, 0.5 mM NADH, 0.2 μ l PK/LDH, and 10 μ l of purified recombinase, with a final reaction capacity of 100 μ l;
s24, FK activity detection experiment: firstly, selecting two FKs with different concentrations to detect fructose, setting 12 concentration gradients in a fructose concentration range of 0-10 mM in a system, starting to double-dilute from the highest concentration of 250 mM (the final concentration in the reaction system is 10 mM) and setting the fructose concentration to be 0mM as a blank control, starting to continuously measure reaction by adding substrates with different concentrations in a Clariostar multifunctional enzyme-linked immunosorbent assay instrument, and measuring the activity by tracking the change of absorbance at 340nm, which corresponds to the conversion of NADH to NAD +, wherein the measurement is carried out for 30 minutes at 25 ℃, inputting corresponding values of detection results of the fructose with different concentrations into Graphpad Prism software for analysis, and analyzing by using regression line fitting of a linear equation to determine the average value +/-standard error (n = 4);
step three, taking substitutes of blood, urine and beverage as examples to detect the fructose of the standard substance, and further detecting the fructose content of the sample:
s3, an experimental pretreatment method and an enzymatic detection method by using the substitute of blood, urine and beverage as a fructose solvent.
3. The fructose detection method based on the fructokinase reaction system as claimed in claim 2, which is characterized in that: the step S12 includes the following steps:
firstly, transforming into an expression bacterium:
adding pET-28a-FK plasmid into BL21 (DE 3) competence for mixing, and then transforming according to the conventional process;
secondly, inducing expression in batches:
and selecting a single colony for amplification and batch expression, wherein the antibiotic is kanamycin, and the single colony is induced by IPTG.
4. The fructose detection method based on the fructokinase reaction system as claimed in claim 2, which is characterized in that: purification was performed by standard methods of His-tag Purification Resin, and protein purity was checked by SDS-PAGE electrophoresis.
5. The fructose detection method based on the fructokinase reaction system as claimed in claim 2, which is characterized in that: the detection of the content of fructose in serum of blood in the step S3 comprises the following steps:
firstly, sample pretreatment: for detecting the fructose content in blood, FBS (fetal bovine serum) and plasma analogue (r-SBFA) are used as solvents to dissolve fructose with different concentrations, 45 mu l of the plasma analogue (r-SBFA) is added into a 1.5 ml EP tube, 5 mu l of fructose with different concentrations and the plasma analogue (r-SBFA) is added, 150 mu l of methanol solution precooled at 4 ℃ is added into each tube, vortex and evenly mixed for 5min, the mixture is placed at-20 ℃ for 60 min and then centrifuged for 13000g (4 ℃, 15 min), supernatant is taken and added into an FK reaction system, and the fructose concentration is detected;
secondly, establishing a standard curve: in the supernatant of the pretreated sample, the highest concentration of fructose is set to be 100 mM, the final concentration in the diluted reaction system is 5 mM, 12 concentration gradients are set, dilution is carried out by multiple times from the highest concentration, and the fructose concentration is set to be 0mM as blank control;
thirdly, sample detection: three samples (n = 3) of different concentrations were chosen to test the accuracy of this protocol.
6. The fructose detection method based on the fructokinase reaction system as claimed in claim 2, which is characterized in that: the detection of the fructose content in the urine in the step S3 comprises the following steps:
firstly, preparing artificial urine (MP-AU): the relevant reagents were added to 100ml of ultrapure water in the following order;
Na2SO4 11.965 mM 0.17 g/100ml
C5H4N4O3 1.487 mM 0.025 g/100ml
Na3C6H5O7·2H2O 2.45 mM 0.072 g/100ml
C4H7N3O 7.791 mM 0.0881 g/100ml
CH4N2O 249.75 mM 1.5 g/100ml
KCl 30.953 mM 0.2308 g/100ml
NaCl 30.053 mM 0.1756 g/100ml
CaCl2·2H2O 1.663 mM 0.0244 g/100ml
NH4Cl 23.667 mM 0.1266 g/100ml
K2C2O4 0.19 mM 0.0035 g/100ml
MgSO4 4.389 mM 0.0449 g/100ml
NaH2PO4·2H2O 18.667 mM 0.224 g/100ml
Na2HPO4·2H2O 4.667 mM 0.1653 g/100ml
secondly, establishing a standard curve: weighing a proper amount of fructose to be dissolved in artificial urine (MP-AU), setting the highest concentration of the fructose to be 100 mM, setting the final concentration in a diluted reaction system to be 5 mM, setting 12 concentration gradients, starting to dilute by multiple times from the highest concentration and setting the concentration of the fructose to be 0mM as blank control;
thirdly, sample detection: three samples (n = 3) at different concentrations were chosen to test the accuracy of this protocol.
7. The fructose detection method based on the fructokinase reaction system as claimed in claim 2, which is characterized in that: the detection of the fructose content in the beverage in the step S3 comprises the following steps:
firstly, establishing a standard curve: weighing a proper amount of fructose, dissolving the fructose in an Ultra-pure water, setting the highest concentration of the fructose to be 100 mM, setting the final concentration in a diluted reaction system to be 5 mM, setting 12 concentration gradients, diluting by multiple proportions from the highest concentration, and setting the concentration of the fructose to be 0mM as a blank reference;
secondly, sample detection: three samples with different concentrations are selected to detect the accuracy of the experimental scheme.
CN202210395204.4A 2022-04-15 2022-04-15 Fructose detection method based on fructokinase reaction system Pending CN114480571A (en)

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