CN117247913A - Extraction method of glucose-6-phosphate dehydrogenase and glucose determination kit - Google Patents
Extraction method of glucose-6-phosphate dehydrogenase and glucose determination kit Download PDFInfo
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- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 title claims abstract description 65
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- IJOJIVNDFQSGAB-SQOUGZDYSA-N 6-O-phosphono-D-glucono-1,5-lactone Chemical compound O[C@H]1[C@H](O)[C@@H](COP(O)(O)=O)OC(=O)[C@@H]1O IJOJIVNDFQSGAB-SQOUGZDYSA-N 0.000 description 1
- 241001155433 Centrarchus macropterus Species 0.000 description 1
- NBSCHQHZLSJFNQ-GASJEMHNSA-N D-Glucose 6-phosphate Chemical compound OC1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H](O)[C@H]1O NBSCHQHZLSJFNQ-GASJEMHNSA-N 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- VFRROHXSMXFLSN-UHFFFAOYSA-N Glc6P Natural products OP(=O)(O)OCC(O)C(O)C(O)C(O)C=O VFRROHXSMXFLSN-UHFFFAOYSA-N 0.000 description 1
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- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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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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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
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- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- 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)
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Abstract
The application provides an extraction method of glucose-6-phosphate dehydrogenase and a glucose determination kit, wherein the extraction method comprises the following steps: activating and then culturing the escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase in an enlarged manner; inducing the bacterial liquid of the expansion culture by adopting an inducer; centrifuging the induced bacterial liquid to obtain bacterial cells; performing ultrasonic crushing on thalli, and taking supernatant to perform protein purification; and dialyzing the purified protein, adding a stabilizer, and freeze-drying to obtain the glucose-6-phosphate dehydrogenase. Compared with the traditional natural tissue extraction process, the method has the characteristics of low cost, high success rate, short time, high yield, easy separation, low pollution and the like; the biosynthesis method can greatly reduce impurities and biotoxicity generated in the natural extraction process of the glucose-6-phosphate dehydrogenase, improve the purity of the enzyme and solve the problem of enzyme source guarantee.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an extraction method of glucose-6-phosphate dehydrogenase and a glucose determination kit.
Background
There are two clinically common methods for determining glucose concentration, one is the glucose oxidase method and the other is the hexokinase method. The hexokinase method for measuring blood sugar has the advantages of higher accuracy and sensitivity, less interference factors and higher specificity. The kit prepared by adopting the hexokinase method comprises hexokinase and glucose-6-phosphate1-dehydrogenase (G6 PDH for short). Under the catalysis of hexokinase, glucose and ATP are subjected to phosphorylation reaction to generate glucose-6-phosphate (G-6-P) and ADP. G-6-P is dehydrogenated under the catalysis of G6PDH to 6-phosphogluconolactone, and NADP is reduced to NADPH (reduced coenzyme II). The rate of NADPH production was proportional to the glucose concentration, and the rate of increase in absorbance was monitored at a wavelength of 340nm to calculate the glucose concentration in serum.
The glucose-6-phosphate dehydrogenase can be obtained from biological tissues, such as yeast, escherichia coli, lactobacillus, flier, streptococcus, and the like, and the enzymatic properties and action mechanisms of the glucose-6-phosphate dehydrogenase from different strains are different. Currently, glucose-6-phosphate dehydrogenase is mainly obtained by two modes of extraction and separation and biosynthesis of natural tissues. However, the extraction and separation mode of the natural tissue has low extraction rate and long time consumption; the existing biosynthesis method has high cost, and the prepared glucose-6-phosphate dehydrogenase has low activity, so that the reagent kit has overlarge addition amount, and is easy to have the phenomena of poor stability, large batch-to-batch difference and the like.
Disclosure of Invention
The invention provides an extraction method of glucose-6-phosphate dehydrogenase and a glucose determination kit, which are used for solving the problems of long time consumption and poor stability in the prior art.
In a first aspect of the present application, there is provided a method for extracting glucose-6-phosphate dehydrogenase, comprising:
activating and then culturing the escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase in an enlarged manner;
inducing the bacterial liquid of the expansion culture by adopting an inducer;
centrifuging the induced bacterial liquid to obtain bacterial cells;
performing ultrasonic crushing on thalli, and taking supernatant to perform protein purification;
and dialyzing the purified protein, adding a stabilizer, and freeze-drying to obtain the glucose-6-phosphate dehydrogenase.
Alternatively, the activated escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase are inoculated into a liquid culture medium containing antibiotics and are cultured overnight at 35-40 ℃ for 16-18 hours, so that the activated escherichia coli glycerol bacteria are obtained.
Optionally, after activating the escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase, mixing the activated bacteria liquid and the liquid culture medium containing antibiotics (5-20) according to the volume ratio of 500, and culturing for 2-3 hours at 35-40 ℃ to obtain the expanded culture bacteria liquid.
Optionally, 1-3 g of antibiotic is contained in each 1000mL of the liquid culture medium;
the antibiotic is selected from one of kanamycin or ampicillin.
Optionally, culturing the bacterial liquid subjected to the expansion culture at 20-30 ℃ for 12-18 hours according to the volume ratio of the bacterial liquid subjected to the expansion culture= (1-10): 1000 as an inducer, so as to obtain the bacterial liquid subjected to the induction; wherein the inducer is one of IPTG or LAC.
Optionally, the thalli are fully dissolved by Tris-HCl-imidazole and then are crushed by ultrasound, and the thalli are centrifugated for 15 to 30 minutes by using 8900 to 10000rpm, and then the supernatant is taken.
Optionally, the purified protein is dialyzed for 6-8 hours by using buffer solution to obtain the dialyzed protein.
Optionally, the stabilizer is selected from at least two of bovine serum albumin, mannitol, trehalose and sucrose.
Optionally, the mass ratio of the stabilizer to the dialyzed protein is (2-4): 100.
In a second aspect of the present application, there is provided a glucose assay kit comprising a glucose-6-phosphate dehydrogenase extracted by the method for extracting a glucose-6-phosphate dehydrogenase provided in any one of the first to fourth aspects.
As can be seen from the technical scheme, the application provides the extraction method of the glucose-6-phosphate dehydrogenase and the glucose determination kit, and compared with the traditional natural tissue extraction process, the extraction method has the characteristics of low cost, high success rate, short time, high yield, easy separation, low pollution and the like; the biosynthesis method can greatly reduce impurities and biotoxicity generated in the natural extraction process of the glucose-6-phosphate dehydrogenase, improve the purity of the enzyme and solve the problem of enzyme source guarantee. In addition, the glucose-6-phosphate dehydrogenase extracted by the extraction method has stable enzyme activity, higher unit enzyme activity, lower addition amount in the kit and smaller influence on a reaction system.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention.
The application provides a method for extracting glucose-6-phosphate dehydrogenase, which comprises the following steps:
step 100, the E.coli glycerol bacteria containing glucose-6-phosphate dehydrogenase are activated and then cultured in an enlarged manner.
The application selects one strain of escherichia coli BL21 (DE 3) or BL21plysS to extract glucose-6-phosphate dehydrogenase, and firstly uses the escherichia coli to recombine to obtain recombinant escherichia coli, and the specific method is as follows: cloning the gene of glucose-6-phosphate dehydrogenase into PET28a or PET28b vector by utilizing DNA recombination technology, introducing the gene into escherichia coli BL21 (DE 3) or BL21plysS, and finally obtaining the recombinant escherichia coli with high yield of glucose-6-phosphate dehydrogenase.
The escherichia coli BL21 (DE 3) glycerinum is prepared by collecting BL21 (DE 3) growing in logarithmic phase and adding bacteria frozen stock solution containing glycerol, and can be directly plated or cultivated in small quantity and large quantity. The glycerol bacteria can be used without complete melting, and the glycerol bacteria can be used after being dipped with a small amount of coating plates or liquid culture.
In one mode, the activated escherichia coli glycerol bacteria are obtained by inoculating escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase into a liquid culture medium containing antibiotics and culturing overnight at 35-40 ℃ for 16-18 hours.
In one possible mode, after the glycerol bacteria of the escherichia coli containing glucose-6-phosphate dehydrogenase are activated, the liquid culture medium containing antibiotics (5-20) and 500 are mixed according to the volume ratio of the activated bacteria liquid to the liquid culture medium containing antibiotics, and the mixture is cultured for 2-3 hours at the temperature of 35-40 ℃ to obtain the bacteria liquid for the expansion culture.
Optionally, 1-3 g of antibiotic is contained in each 1000mL of the liquid culture medium; wherein the antibiotic is selected from one of kanamycin or ampicillin.
In the present application, the liquid medium is LB liquid medium.
Step 200, inducing the bacterial liquid of the expansion culture by adopting an inducer.
In the step, the bacterial liquid subjected to the expansion culture is subjected to overnight culture for 12-18 hours at 20-30 ℃ according to the volume ratio of the inducer to the bacterial liquid subjected to the expansion culture= (1-10): 1000, so as to obtain the bacterial liquid after the induction.
The choice of the inducer and the conditions of induction are critical for the stable expression of glucose-6 phosphate dehydrogenase. In the application, the inducer is one of IPTG or LAC, and the concentration of the inducer is 1M. The volume ratio of the inducer to the bacterial liquid for the expansion culture is required to be within a selected range, if the addition amount of the inducer is too small, the nutrient substances in the culture medium are insufficient, and the quality of the culture medium is affected; if the inducer is added in an excessive amount, the nutrient substances in the culture medium are excessive, and the quality of the culture medium is also affected. The temperature and time of the culture are selected according to the different culture objects and inducers, and the induction effect is best under the culture conditions of the application, so that the stably expressed glucose-6 phosphate dehydrogenase can be obtained.
Before induction, detecting the bacterial liquid after the expansion culture, in one realizable mode, taking 2-3 ml bacterial liquid after the expansion culture, detecting the bacterial liquid with an ultraviolet-visible spectrophotometer to have an OD600 value of more than or equal to 0.5, and then adding an inducer.
And 300, centrifuging the induced bacterial liquid to obtain bacterial cells.
In this step, centrifugation was directly performed to remove the supernatant, thereby obtaining a cell.
Step 400, performing ultrasonic disruption on the thalli, and taking supernatant for protein purification.
In the step, the thalli are fully dissolved by Tris-HCl-imidazole and then are crushed by ultrasound, and are centrifuged for 15 to 30 minutes by using 8900 to 10000rpm, and then the supernatant is taken.
The concentration of Tris-HCl-imidazole used in the present application was 20mM.
And 500, dialyzing the purified protein, adding a stabilizer, and freeze-drying to obtain the glucose-6-phosphate dehydrogenase.
In the step, ni column purification is adopted, and the purified protein is subjected to dialysis treatment by using buffer solution for 6-8 hours, so that the dialyzed protein is obtained. In this application, 20mM Tris-0.15MNaCl or phosphate was selected as buffer, and the pH was 7.0-8.0.
The stabilizer is at least two selected from bovine serum albumin, mannitol, trehalose and sucrose. Optionally, the mass ratio of the stabilizer to the dialyzed protein is (2-4): 100.
The protein sample added with the stabilizer is freeze-dried for 24-48 hours by using an ultralow temperature freeze dryer, freeze-dried powder is collected, and the quality of the freeze-dried powder is weighed and stored in a refrigerator at the temperature of minus 20 ℃.
In order to prolong the preservation time, the preservative is also added before freeze-drying, and the mass ratio of the preservative to the protein sample is (0.1-0.2): 100. The preservative can be sodium azide or other substances with the same function as the sodium azide.
The extraction and separation of the natural tissues requires a continuous purification process, and the time consumption is long; the purification process has unnecessary loss of target protein, resulting in low extraction rate. The extraction method provided by the application can be completed only by Ni column purification, and has high efficiency and higher purity of target protein.
The application also provides a glucose determination kit which is prepared by adopting the glucose-6-phosphate dehydrogenase extracted by the extraction method of the glucose-6-phosphate dehydrogenase.
The application provides a glucose assay kit includes: r1 reagent and R2 reagent. Wherein the R1 reagent comprises 20-50mM Tris-HCl buffer solution, 0.01-0.05% of surfactant, 1-2% of stabilizer and 0.05-0.1% of preservative; the R2 reagent comprises Tris-HCl buffer solution, glucose-6-phosphate dehydrogenase 5-10KU, hexokinase 4.5-8KU and preservative 0.05-0.1%.
Optionally, the Tris-HCl buffer solution is Tris-HCl buffer solution with pH value of 6.5-8.
Optionally, the surfactant is one of tween 20 or triton x-100.
Optionally, the stabilizer is one of bovine serum albumin, trehalose, mannitol and sucrose.
Optionally, the preservative is one of sodium azide or PC 300.
The activity of glucose-6-phosphate dehydrogenase was evaluated by detecting glucose with the above kit, and the measurement parameters were as follows:
the present application is further illustrated by the following specific examples.
Example 1：
Extracting glucose-6-phosphate dehydrogenase according to the following steps:
(1) Preparing LB liquid medium containing kanamycin, adding 1g kanamycin into 1000mLLB liquid medium, and mixing thoroughly;
(2) Activating: glycerol bacteria cryopreserved at-80 ℃ are inoculated into 50mL of LB liquid medium containing kanamycin by using an L-shaped coating rod, and cultured in a shaking table at 35 ℃ and 180rpm for 18 hours;
(3) And (3) performing expansion culture: inoculating 10ml of overnight culture bacteria liquid into 500ml of LB liquid medium containing kanamycin, and culturing for 2h at 37 ℃ and 180 rpm;
(4) Induction: taking 3ml of bacterial liquid, adopting an ultraviolet-visible spectrophotometer to detect that the OD600 value is more than or equal to 0.5, adding 500uL1MIPTG into the bacterial liquid subjected to expansion culture in an ultra-clean workbench, and culturing at 25 ℃ at 180rpm for 12 hours;
(5) And (5) bacterial liquid centrifugation: the bacterial liquid after overnight culture is split and balanced by adopting a centrifugal bottle and then centrifuged for 15min at 4 ℃;
(6) And (3) thallus treatment: sufficiently dissolving thalli by adopting a 20mM Tris-HCl imidazole solution (pH=8.0), performing ultrasonic crushing for 30min, centrifuging the crushed bacterial liquid in a low-temperature refrigerated centrifuge at 8900rpm for 30min, and taking a supernatant for protein purification;
(7) Protein purification:
(1) adopting an HD-3000 nucleic acid protein detection system to purify the protein, adopting a peristaltic pump to flush the Ni column at a pump speed of 3ml/min, wherein the flushing amount is about 10 times of the column volume;
(2) column equilibration (about 10 column volumes) was performed by preparing 20mM Tris-HCl-imidazole equilibration buffer at a pump speed of 3 ml/min; after the curve is stable, the light quantity is adjusted to 100, the knob is turned to 0.2A, the light quantity is adjusted to about 0-5, and sample loading is started;
(3) performing column treatment on a sample filtered by a 0.22 micrometer filter membrane, pumping at a speed of 2ml/min, and collecting flow-through liquid;
(4) performing system liquid elution by adopting 20mM Tris-HCl-imidazole, performing gradient elution when the light quantity is close to 0', and performing post-treatment and sealing up of the Ni column by using alcohol after the elution is finished;
(8) Sample dialysis: dialyzing the eluted protein with dialysis buffer solution 20mM Tris-HCl,0.15MNaClPH8.0 for 8 hr, weighing sample mass after dialysis, adding stabilizer 2% bovine serum albumin and 1% mannitol, and preserving in refrigerator at-80deg.C after antiseptic 0.1% sodium azide;
(9) Sample lyophilization: and freeze-drying the sample for 48 hours by adopting a freeze dryer, collecting freeze-dried powder, weighing the mass of the dry powder, and storing the dry powder in a refrigerator at the temperature of minus 20 ℃.
A glucose assay kit was prepared according to the following steps:
configuration of R1 reagent: 2.4228g of 20mM Tris is dissolved in 1L of distilled water, the PH is adjusted to 8.0 by using hydrochloric acid, 1mL of Tween 20 is added for mixing, 100g of trehalose is added for mixing, and 1g of sodium azide is added for mixing, thus obtaining the R1 reagent.
Configuration of R2 reagent: 2.4228g of 10mM Tris is dissolved in 1L of distilled water, the PH is regulated to 8.0 by using hydrochloric acid, 1g of sodium azide is added for uniform mixing, 5KU glucose-6-phosphate dehydrogenase is added, 4.5KU hexokinase is added for uniform mixing, and the R2 reagent is obtained.
Example 2：
Extracting glucose-6-phosphate dehydrogenase according to the following steps:
(1) Preparing LB liquid medium containing kanamycin, adding 2g kanamycin into 1000mLLB liquid medium, and mixing thoroughly;
(2) Activating: glycerol bacteria cryopreserved at-80 ℃ were inoculated into 50mL of LB liquid medium containing kanamycin using L-shaped coating bars, and cultured overnight in a shaker at 36 ℃ at 180rpm for 17h;
(3) And (3) performing expansion culture: inoculating 5ml of overnight culture bacteria liquid into 500ml of LB liquid medium containing kanamycin, and culturing at 35 ℃ for 3h at 180 rpm;
(4) Induction: 3ml of bacterial liquid is taken, after the OD600 value is more than or equal to 0.5 detected by an ultraviolet-visible spectrophotometer, 5ml of 1MLAC is added into the bacterial liquid for expansion culture in an ultra-clean workbench, and the bacterial liquid is cultured for 18 hours at 20 ℃ at 180 rpm;
(5) And (5) bacterial liquid centrifugation: the bacterial liquid after overnight culture is split and balanced by adopting a centrifugal bottle and then centrifuged for 15min at 4 ℃;
(6) And (3) thallus treatment: sufficiently dissolving the thalli by adopting a 20mM Tris-HCl imidazole solution (pH=8.0), performing ultrasonic crushing for 30min, centrifuging the crushed bacterial liquid in a low-temperature refrigerated centrifuge at 9500rpm for 25min, and taking a supernatant for protein purification;
(7) Protein purification:
(1) adopting an HD-3000 nucleic acid protein detection system to purify the protein, adopting a peristaltic pump to flush the Ni column at a pump speed of 3ml/min, wherein the flushing amount is about 10 times of the column volume;
(2) column equilibration (about 10 column volumes) was performed by preparing 20mM Tris-HCl-imidazole equilibration buffer at a pump speed of 3 ml/min; after the curve is stable, the light quantity is adjusted to 100, the knob is turned to 0.2A, the light quantity is adjusted to about 0-5, and sample loading is started;
(3) performing column treatment on a sample filtered by a 0.22 micrometer filter membrane, pumping at a speed of 2ml/min, and collecting flow-through liquid;
(4) performing system liquid elution by adopting 20mM Tris-HCl-imidazole, performing gradient elution when the light quantity is close to 0', and performing post-treatment and sealing up of the Ni column by using alcohol after the elution is finished;
(8) Sample dialysis: dialyzing the eluted protein with dialysis buffer solution 20mM Tris-HCl0.15MNaClPH8.0 for 7h, weighing the mass of the sample after the dialysis is finished, adding stabilizer 1% bovine serum albumin and 1% trehalose, and preserving in a refrigerator at-80 ℃ after preservative 0.2% sodium azide;
(9) Sample lyophilization: and freeze-drying the sample for 48 hours by adopting a freeze dryer, collecting freeze-dried powder, weighing the mass of the dry powder, and storing the dry powder in a refrigerator at the temperature of minus 20 ℃.
A glucose assay kit was prepared according to the following steps:
configuration of R1 reagent: 2.4228g of 20mM Tris is dissolved in 1L of distilled water, the PH is adjusted to 8.0 by using hydrochloric acid, 1mL of Tween 20 is added for mixing, 100g of trehalose is added for mixing, and 1g of sodium azide is added for mixing, thus obtaining the R1 reagent.
Configuration of R2 reagent: 2.4228g of 10mM Tris is dissolved in 1L of distilled water, the PH is regulated to 8.0 by using hydrochloric acid, 1g of sodium azide is added for uniform mixing, 5KU glucose-6-phosphate dehydrogenase is added, 4.5KU hexokinase is added for uniform mixing, and the R2 reagent is obtained.
Example 3：
Extracting glucose-6-phosphate dehydrogenase according to the following steps:
(1) Preparing LB liquid medium containing kanamycin, adding 3g of ampicillin into 1000mLLB liquid medium, and fully mixing;
(2) Activating: glycerol bacteria cryopreserved at-80 ℃ are inoculated into 50mL of LB liquid medium containing kanamycin by using an L-shaped coating rod, and cultured in a shaking table at 37 ℃ and 180rpm for 16 hours;
(3) And (3) performing expansion culture: inoculating 20ml of overnight culture bacteria liquid into 500ml of LB liquid medium containing kanamycin, and culturing at 40 ℃ for 2.5h at 180 rpm;
(4) Induction: taking 3ml of bacterial liquid, adopting an ultraviolet-visible spectrophotometer to detect that the OD600 value is more than or equal to 0.5, adding 500uL1MIPTG into the bacterial liquid subjected to expansion culture in an ultra-clean workbench, and culturing at 30 ℃ at 180rpm for 12 hours;
(5) And (5) bacterial liquid centrifugation: the bacterial liquid after overnight culture is split and balanced by adopting a centrifugal bottle and then centrifuged for 15min at 4 ℃;
(6) And (3) thallus treatment: fully dissolving thalli by adopting 20mM Tris-HCl imidazole solution (pH=8.0), performing ultrasonic crushing for 30min, centrifuging the crushed bacterial liquid in a low-temperature refrigerated centrifuge at 10000rpm for 15min, and taking a supernatant for protein purification;
(7) Protein purification:
(1) adopting an HD-3000 nucleic acid protein detection system to purify the protein, adopting a peristaltic pump to flush the Ni column at a pump speed of 3ml/min, wherein the flushing amount is about 10 times of the column volume;
(2) column equilibration (about 10 column volumes) was performed by preparing 20mM Tris-HCl-imidazole equilibration buffer at a pump speed of 3 ml/min; after the curve is stable, the light quantity is adjusted to 100, the knob is turned to 0.2A, the light quantity is adjusted to about 0-5, and sample loading is started;
(3) performing column treatment on a sample filtered by a 0.22 micrometer filter membrane, pumping at a speed of 2ml/min, and collecting flow-through liquid;
(4) performing system liquid elution by adopting 20mM Tris-HCl-imidazole, performing gradient elution when the light quantity is close to 0', and performing post-treatment and sealing up of the Ni column by using alcohol after the elution is finished;
(8) Sample dialysis: dialyzing the eluted protein with dialysis buffer solution 20mM Tris-HCl0.15MNaClPH8.0 for 6h, weighing the mass of the sample after the dialysis is finished, adding stabilizer 2% sucrose and 2% mannitol, and preserving in a refrigerator at-80 ℃ after preservative 0.1% sodium azide;
(9) Sample lyophilization: and freeze-drying the sample for 48 hours by adopting a freeze dryer, collecting freeze-dried powder, weighing the mass of the dry powder, and storing the dry powder in a refrigerator at the temperature of minus 20 ℃.
A glucose assay kit was prepared according to the following steps:
configuration of R1 reagent: 2.4228g of 20mM Tris is dissolved in 1L of distilled water, the PH is adjusted to 8.0 by using hydrochloric acid, 1mL of Tween 20 is added for mixing, 100g of trehalose is added for mixing, and 1g of sodium azide is added for mixing, thus obtaining the R1 reagent.
Configuration of R2 reagent: 2.4228g of 10mM Tris is dissolved in 1L of distilled water, the PH is regulated to 8.0 by using hydrochloric acid, 1g of sodium azide is added for uniform mixing, 5KU glucose-6-phosphate dehydrogenase is added, 4.5KU hexokinase is added for uniform mixing, and the R2 reagent is obtained.
Comparative example：
Glucose assay kit (hexokinase method) manufactured by Jilin-based egg biotechnology Co., ltd; lot number: 20220910. the method of preparing R1 and R2 in the kit was the same as in example 1, except that glucose-6-phosphate dehydrogenase was purchased to Roche.
The detection method comprises the following steps:
by using a Hitachi 7100 biochemical analyzer, 10 clinical samples with different concentrations are taken for detection, and the relevance and the like of the kit provided by the application are compared according to the detection result of blood values.
Table 1 results of the tests of the respective examples and comparative examples
TABLE 2 detection results after heat treatment of example 1
TABLE 3 detection results after heat treatment of example 2
TABLE 4 detection results after heat treatment of example 3
The results in Table 1 show that the kit prepared by the extracted glucose-6-phosphate dehydrogenase has better contrast correlation with a glucose determination kit (hexokinase method) of Jilin-based egg biotechnology Co., ltd, consistent measured value and a correlation coefficient of more than 0.99; the results in tables 2,3 and 4 show that the enzyme activity stability is about 2 years and the enzyme performance is stable through high temperature test.
In the comparative example, glucose-6-phosphate dehydrogenase was purchased from Roche, and the glucose-6-phosphate dehydrogenase extracted according to the present invention was compared with Roche glucose-6-phosphate dehydrogenase in terms of unit enzyme activity.
TABLE 5 example 1 and comparative example unit enzyme Activity
| R2/L | Quality (g) of glucose-6-phosphate dehydrogenase | Total enzyme activity (KU) | Unit enzyme activity (KU/g) |
| Example 1 | 0.053 | 5 | 94.34 |
| Example 2 | 0.062 | 5 | 80.65 |
| Example 3 | 0.069 | 5 | 72.46 |
| Comparative example | 0.084 | 5 | 59.52 |
The results in Table 5 show that the unit enzyme activity of the glucose-6-phosphate dehydrogenase extracted by the invention is superior to that of the glucose-6-phosphate dehydrogenase of Roche, the addition amount in the kit is lower, and the influence on the reaction system is smaller.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as providing for the use of additional embodiments and advantages of all such modifications, equivalents, improvements and similar to the present invention are intended to be included within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A method for extracting glucose-6-phosphate dehydrogenase, comprising:
activating and then culturing the escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase in an enlarged manner;
inducing the bacterial liquid of the expansion culture by adopting an inducer;
centrifuging the induced bacterial liquid to obtain bacterial cells;
performing ultrasonic crushing on thalli, and taking supernatant to perform protein purification;
and dialyzing the purified protein, adding a stabilizer, and freeze-drying to obtain the glucose-6-phosphate dehydrogenase.
2. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the activated escherichia coli glycerol bacteria are obtained by inoculating escherichia coli glycerol bacteria containing glucose-6-phosphate dehydrogenase into a liquid culture medium containing antibiotics and culturing overnight at 35-40 ℃ for 16-18 hours.
3. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein after activating the E.coli glycerol bacteria containing glucose-6-phosphate dehydrogenase, the liquid culture medium= (5-20) containing antibiotics is mixed according to the volume ratio of 500 to the activated bacteria liquid, and the mixture is cultured at 35-40 ℃ for 2-3 hours to obtain the expanded culture bacteria liquid.
4. The method for extracting glucose-6-phosphate dehydrogenase according to claim 2 or 3, wherein 1 to 3g of the antibiotic is contained per 1000mL of the liquid medium;
the antibiotic is selected from one of kanamycin or ampicillin.
5. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the bacterial liquid subjected to the expansion culture is subjected to overnight culture at 20-30 ℃ for 12-18 hours according to the volume ratio of an inducer, namely bacterial liquid subjected to the expansion culture= (1-10): 1000, so as to obtain an induced bacterial liquid; wherein the inducer is one of IPTG or LAC.
6. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the cells are sufficiently dissolved in Tris-HCl-imidazole, then subjected to ultrasonic disruption, and centrifuged at 8900-10000 rpm for 15-30 min, and then the supernatant is obtained.
7. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the purified protein is subjected to dialysis treatment with a buffer solution for 6 to 8 hours to obtain a dialyzed protein.
8. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the stabilizer is at least two selected from the group consisting of bovine serum albumin, mannitol, trehalose and sucrose.
9. The method for extracting glucose-6-phosphate dehydrogenase according to claim 1, wherein the mass ratio of the stabilizer to the dialyzed protein is (2-4): 100.
10. A glucose assay kit comprising a glucose-6-phosphate dehydrogenase extracted by the method for extracting a glucose-6-phosphate dehydrogenase according to any one of claims 1 to 9.
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