CN107119059B

CN107119059B - Xanthine oxidase for clinical detection, coding gene thereof and application thereof

Info

Publication number: CN107119059B
Application number: CN201710262579.2A
Authority: CN
Inventors: 闫达中
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2017-04-20
Filing date: 2017-04-20
Publication date: 2020-03-20
Anticipated expiration: 2037-04-20
Also published as: CN107119059A

Abstract

The invention relates to a xanthine oxidase gene, the sequence of which is SEQ ID NO 1 or a synonymous mutant thereof; also relates to a xanthine oxidase expression vector with high GC content; also relates to the xanthine oxidase coded by the xanthine oxidase gene and application thereof; also relates to a method for oxidizing hypoxanthine and xanthine.

Description

Xanthine oxidase for clinical detection, coding gene thereof and application thereof

Technical Field

The invention relates to the field of molecular biology, in particular to a xanthine oxidase gene, a xanthine oxidase coded by the xanthine oxidase gene, application of the xanthine oxidase gene, a xanthine oxidase gene expression vector, an expression strain and a method for oxidizing hypoxanthine and xanthine.

Background

Xanthine oxidase (Xanthine oxidase, abbreviated as XOD, EC 1.1.3.22) is widely present in tissues and cells of various organisms, is a cytoplasmic complex enzyme, is a key enzyme for purine metabolism of organisms, and belongs to a molybdenum enzyme family. At O₂In the presence of XOD, molecular oxygen is used as a direct electron acceptor, and the oxidation reaction can be catalyzed without coenzyme. XOD is involved in almost all biochemical processes of cells, nucleotides are first degraded in vivo into nucleosides and phosphates, and nucleosides are decomposed into purine bases, pyrimidine bases and ribose under the action of nucleosidase. XOD catalyzes the oxidation of hypoxanthine and xanthine to uric acid and subsequent metabolism to urea according to the following equation:

in the reaction formula, (1) represents xanthine oxidase, (2) represents urate oxidase, (3) represents allantoin enzyme, and (4) represents allantoin enzyme

In the metabolic process, substances having important physiological functions such as superoxide or oxygen radicals are also produced, and are drawing attention in close relation to diseases such as ischemic reperfusion injury, hyperuricemia, gout, rheumatism, hepatitis, and arthritis.

At present, XOD has been developed into a commercial enzyme with a higher added value, and can be clinically used for preparing immune antibodies, tumor suppressors and detection and diagnosis reagents for diseases, mainly used for detecting xanthine, hypoxanthine and inosine (inosine), assisting in detecting guanine and guanosine, and also used for determining the activity of 5' -nucleoside phosphorylase, and is suitable for detecting hepatobiliary tumors. XOD is also widely used for detecting serum inorganic phosphorus level (hyperphosphatemia) and superoxide dismutase SOD activity. Therefore, the method has important theoretical significance and wide application prospect in the development and research of the xanthine oxidase XOD.

In addition, the content of hypoxanthine in the body of fish increases as its putrescence increases, so by measuring the change in hypoxanthine in the body, the freshness of fish can be determined. The xanthine oxidase can also be used for degrading carotenoid to prepare tobacco flavor.

The xanthine oxidase used at home and abroad at present is mainly obtained from milk. The source of the xanthine oxidase is single, the production cost is high, and the content and the quality of the xanthine oxidase are influenced by different regions and different types of cows, so that the xanthine oxidase is greatly limited in application.

Therefore, a novel xanthine oxidase gene and expression system are required.

Disclosure of Invention

In order to solve the above problems, the present invention provides a xanthine oxidase gene, the sequence of which is SEQ ID NO. 1 or a synonymous mutant thereof.

The invention also provides an expression vector of the xanthine oxidase, which comprises a xanthine oxidase expression cassette, wherein the xanthine oxidase expression cassette consists of a promoter and a xanthine oxidase gene connected to the downstream of the promoter, and the sequence of the xanthine oxidase gene is SEQ ID NO. 1 or a synonymous mutant thereof.

In a preferred embodiment, the promoter is an inducible promoter.

The invention also provides a recombinant strain for expressing the xanthine oxidase, which is obtained by transforming the expression vector of the xanthine oxidase gene into an expression host.

The invention also provides xanthine oxidase which is coded by the xanthine oxidase gene.

The invention also provides application of the xanthine oxidase in preparation of a reagent for detecting hypoxanthine and/or xanthine concentration.

The present invention also provides a method for oxidizing hypoxanthine and/or xanthine in a solution, comprising the step of adding to said solution the xanthine oxidase described above.

In a preferred embodiment, the pH of the solution is 6.0 to 9.0.

In another preferred embodiment, the temperature of the solution is from 25 to 55 ℃.

In another preferred embodiment, the solution is free of metal ions, or contains Mg²⁺、Ca²⁺、Mo⁶⁺、Co²⁺Or Mn²⁺。

Drawings

Fig. 1 is an electrophoretogram of extracted YF06 genome;

FIG. 2 is an electrophoretogram of PCR product obtained by PCR amplification using YF06 genome as template and

primers

1 and 2;

FIG. 3 is an electrophoresis diagram of EcoRI and HindIII double digestion detection recombinant plasmid pGEMT-xodAB-His;

FIG. 4 is an electrophoresis diagram of EcoRI and Hind III double digestion detection of recombinant plasmid pVLT 33-xodAB-His;

FIG. 5 is a XOD purification plot showing protein elution peak times;

FIG. 6 is an SDS-PAGE of pAW340[ pVLT33] and pAW340[ pVLT33-xodAB-His ] crude enzyme solutions purified of

proteins

7, 8 and 9 collected,

lanes

1,2 and 3 showing the higher concentrations of

proteins

7, 8 and 9 collected after purification; lane 4 is crude enzyme solution before purification; lane 5 is control pAW340[ pVLT33 ]; m is a protein molecule marker;

FIG. 7 is a graph of a concentration standard curve prepared with BSA;

FIG. 8 is a pH-XOD viability curve;

FIG. 9 is a graph showing the enzyme activity curves of XOD after 16h at different pH values;

FIG. 10 is a graph of viability at temperature versus XOD;

FIG. 11 is a graph showing the activity of XOD after 30min at different temperatures;

FIG. 12 is a double reciprocal plot of the kinetics of the enzymatic reaction of XOD to catalyze the oxidation of xanthines.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

1. Screening of XOD-producing strains

Collecting activated sludge in sewage treatment pools of various beer production enterprises, fully and uniformly mixing, respectively taking a small amount of samples, putting the samples into a clean and sterilized triangular flask, adding a proper amount of sterile water, performing shake culture on a shaking table for 2 days to fully release thalli in a soil sample, and centrifuging to take supernatant to obtain mother liquor containing microorganisms in the soil sample.

Adding 1mL of the mother solution into 50mL of the selection medium, and culturing for 7 days at 30 ℃ under shaking (180 r/min); then continuously subculturing for 4 times, taking 200 mu L of culture solution for gradient dilution, coating a selective culture medium plate, and culturing for 7 days at 30 ℃; and (3) when a single colony grows out, selecting the single colony to a solid inorganic salt selective culture medium, culturing at constant temperature of 30 ℃, selecting a colony with a larger hydrolysis ring, obtaining an arthrobacter strain YF06 in the screening process, and performing further identification.

2. Extracting XOD from YF06

1) Inoculating YF06 into sterilized 1/4 nutrient medium, culturing at 170r/min and 30 deg.C for 24 h;

2) each of the four flasks contained 100mL of 1/4 nutrient medium was added with 1mL of the culture medium, and after culturing for 48 hours, the OD was measured₆₀₀The value is 0.6-0.8, and xanthine is added for induction for 24 hours;

3) centrifuging the above 400mL culture at 8000r/min for 10min, discarding supernatant to obtain precipitate, suspending the precipitate with 15mL50mM Tris-HCl (pH 7.5), and performing ultrasonic cell disruption at 0 deg.C and ultrasonic power of 400w and ultrasonic frequency of 2s at an interval of 2s for 25 min. After the crushing is finished, centrifuging for 15min at 0 ℃ and 12000rpm, and removing cell fragments to obtain cell crushing liquid supernatant;

4) the obtained supernatant was added slowly with ammonium sulfate solid at 0 ℃ until saturation became 45%, the mixture was stirred while adding, salting out was carried out overnight, centrifugation was carried out at 8000rpm for 15min, the supernatant was removed, a minimum volume of enzyme buffer Tris-HCI buffer (50mM, pH 7.5) was added to the precipitate to completely dissolve it, and the obtained solution was determined to have XOD enzyme activity, and it was found that XOD enzyme-encoding gene was likely to be present in the genome of YF 06.

3. Isolation of XOD enzyme encoding Gene from Strain YFO6

Extracting YF06 genome with Premega genome extracting kit, and gel electrophoresis detecting small amount of DNA. The results are shown in FIG. 1, where the extracted genome is relatively complete. For a single type of DNA sample of the same kind, the amount of the nucleic acid dye embedded is proportional to the DNA content of the sample solution, the extracted DNA brightness is about 3 times of that of Marker, and the DNA concentration can be calculated from the amount of the Marker added. Using a micro ultraviolet spectrophotometer to determine the DNA concentration of 5ug/ml and DNAOD₂₆₀/OD₂₈₀The ratio of (A) to (B) was about 1.71, and the purity was proved to be good.

Downloading XOD size subunit gene sequences in an NCBI GenBank database, carrying out multi-sequence comparison by using clustalw2, and designing primers capable of amplifying XOD in upstream and downstream conserved regions of the XOD gene by using Primer5.0 software. The sequences of the primer pairs are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 3.

Primer 1: 5'-GAATTCCATG TGGCCCGCGCTGACCGTCACC-3' (SEQ ID NO: 2);

primer 2: 5'-AAGCTTTCA GTG GTG GTG GTG GTG GTG GCCCATCGGGCCCACGGTGT-3' (SEQ ID NO: 3).

YF06 is used as a template,

primers

1 and 2 are used for PCR amplification, an amplification product is detected by 0.8% agarose gel electrophoresis, the result is shown in figure 2, a band is obviously seen below 5000 and is consistent with the size of an expected target gene, the sequence is shown as SEQ ID NO. 1 after sequencing, the amplification product XOD is used for encoding a gene xodAB, and the GC content in the amplification product XOD is more than 75%.

4. Construction of recombinant expression plasmid pVLT33-xodAB-His

And (2) carrying out agarose gel electrophoresis recovery on the PCR product, connecting the product with A. tailing product to pGEMT-easy vector, transferring into DH5 α, coating on an LB solid plate containing ampicillin, culturing at 37 ℃ for 12h, randomly selecting transformant, inoculating the transformant into 3mL of liquid LB culture medium, extracting plasmid by using an alkaline lysis method after 12h at 37 ℃, and carrying out enzyme digestion to identify the transformant, wherein the result is shown in figure 3, the size of the pGEMT-easy vector is 2867bp, the size of the xodAB gene is 3597bp, two bands are observed behind the plasmid extracted by enzyme digestion and are matched with the sizes of the pGEMT-easy vector and the gene xodAB, therefore, the xodAB is successfully cloned to the pGEM-T vector, is sent to the marine engineering for sequencing, and the correct recombinant plasmid is named as pGEMT-xodAB.

The correct transformants were plated, a single colony was picked and inoculated on LB (ampicillin-containing) for 12h at 37 ℃, plasmids were extracted with the kit, and after double digestion, gel electrophoresis was performed to recover xodAB gene fragments, the recovered target gene was ligated to pVLT33 vector recovered by EcoRI/HindIII double digestion, and transformed into E.coli DH5 α, which was spread on a solid LB (kanamycin 50. mu.g/mL) plate, and after 12h of culture, a single colony was randomly picked and inoculated on 5mL of liquid LB (kanamycin, 37 ℃, 12h after boiling to extract plasmids, and transformants were identified by double cutting with EcoRI and HindIII, as shown in FIG. 4, pVLT33 vector was 9.7kb in size, XOD gene xodAB was 3597bp in size, and from this figure, two bands after digestion of the plasmid were observed, which coincided with vector pVLT33 and gene xodAB, and thus, xodAB was successfully cloned on vector VLT 33.

5. Construction of expression Strain

And (3) coating a plate with a correctly identified transformant, selecting a single colony, extracting a plasmid by a boiling method, and transferring the recombinant plasmid into pAW340 to obtain the expression strain.

Centrifuging the induced bacterial liquid, performing ultrasonic disruption, centrifuging at 4 ℃ and 12000rpm for 15min, and removing cell debris to obtain the crude cell enzyme liquid. And (4) taking the crude enzyme liquid of the cells to measure the enzyme activity, and measuring that the shake flask fermentation yield of the engineering bacterium enzyme is 1.52U/mL. The obtained re-engineered strain was found to be active XOD.

6. Purification of xanthine oxidase

The crude enzyme solution of recombinant engineered bacterium pAW340[ pVLT33-xodAB-His ] for inducing expression is added with imidazole (0.005M) and NaCl (0.5M) for filtration, and then protein is purified by His Trap nickel column. During the collection of the purified enzyme, starting from the increase in the volume of solution B, one tube was collected per 1 mL. Purification as shown in FIG. 5, it can be seen that No. 5 to No. 10 represent the time periods of peak appearance, and no miscellaneous peaks appear. Taking pAW340[ pVLT33] and pAW340[ pVLT33-xodAB-His ] crude enzyme solutions and 20 μ L of purified and collected proteins No. 7, 8 and 9, adding 5 Xloading buffer solution, boiling in boiling water for 10min, centrifuging, taking supernatant, and performing SDS-PAGE gel detection, wherein the results are shown in figure 6, and

lanes

1,2 and 3 are proteins No. 7, 8 and 9 with higher concentration collected after purification; lane 4 is crude enzyme solution before purification; lane 5 is control pAW340[ pVLT33 ]; m is protein molecular weight marker.

After collection, the activity of each tube is measured, and No. 7, No. 8 and No. 9 with higher activity are selected to be centrifuged for 3min at 7000 rpm of an ultrafiltration centrifuge tube. The purified enzyme solution was stored in two tubes.

The absorbance at 595nm was measured using BSA standard solutions at different concentrations and a standard curve was plotted, as shown in FIG. 7, for BSA standards at concentrations ranging from 10-50. mu.g/mL and A₅₉₅The standard curve equation is that y is 0.012x +0.008, R is²0.993, can be used for protein concentration calculation. pAW340[ pVLT33-xodAB-His ] after purification was calculated from a standard curve]The concentration of the protein sample of (2) was 26. mu.g/mL.

Determination of XOD enzyme Activity

Xanthine oxidase in the course of catalyzing the reaction of xanthine oxidation, 1mol of oxygen and water will be consumed per 1mol of xanthine oxidized to produce 1mol of uric acid and 1mol of H₂O₂. Uric acid has a maximum absorption peak at 293 nm. The uric acid concentration can be determined by detecting the absorbance of the solution at 293nmAnd thus the XOD enzyme activity is determined by the change in concentration.

The pure enzyme solution was added to the solution containing xanthine and measured at the absorbance at 293nm with an ultraviolet spectrophotometer. The activity of the xanthine oxidase is determined according to the ultraviolet absorption condition of the reaction product uric acid by carrying out detection every 30 s.

1U of enzyme activity is defined as the amount of enzyme required to produce 1. mu. mol of uric acid in one minute is 1U. The enzyme activity determination experiment adopts a method for determining enzyme activity on a product specification, and specifically comprises the following reagents and steps:

A.Tris-HCl buffer,pH 7.5 ：0.1M

B.Sodium hydroxide solution ：0.0025M

C.Xanthine solution ：10mM

D.Oxonic acid potassium salt solution ：1mM

E.Enzyme diluent ：50mM Tris-HCl buffer,pH7.5

adding 2.24ml of solution A, 80 μ L of solution C and 80 μ L of solution D into an ultraviolet quartz cell, gently blowing, bathing at 37 deg.C for 45min, adding crude enzyme solution into the mixed solution, measuring the absorbance change at 293nm in an ultraviolet spectrophotometer, and recording the OD once every 30 seconds₂₉₃And changing and recording data.

According to the calculation formula of enzyme activity provided in Japanese "TOYOBO ENZYMES" document:

Volume activity(U/ml)＝ΔOD/min×2.0×df

Weight activity(U/mg)＝(U/ml)×1/C

wherein the meaning of each symbol or number is as follows: vt is the total volume (2.5 ml); vs is the sample volume (0.1 ml); 12.5 absorption coefficient per millimole of uric acid (cm) under the detection conditions²mu.M; 1.0 is the optical path length (cm); df is the dilution factor; c is the lytic enzyme concentration (mg/ml).

1) Effect of pH on XOD enzyme Activity and stability

Preparing enzyme detection solution with buffer solutions with different pH values (pH 4.0-10.0), measuring enzyme activity according to the method, and determining the optimum pH value of XOD reaction. Diluting the enzyme solution with buffer solutions with different pH values (pH4.0-11.0), keeping the temperature at 25 ℃ for 15h, and then measuring the residual enzyme activity. A curve of pH and enzyme activity was prepared by using the enzyme activity of XOD at the optimum pH, and the pH stability of XOD was examined.

The activity of XOD was measured at 37 ℃ in each of the buffers at pH4.0 to 10.0, and as shown in FIG. 8, the enzyme activity was 0 at pH4.0, and the xanthine oxidase activity gradually increased with increasing pH, reaching a maximum value at pH 8.0. The decrease then started and the activity disappeared at pH 10.0. Therefore, the enzyme has activity between pH 6 and 9, and the optimum reaction pH of the enzyme is 8.0.

The enzyme solution was added to buffers of different pH values, and after incubation at 25 ℃ for 16 hours, the residual enzyme activity was measured, and the results are shown in FIG. 9, in which the xanthine oxidase had no activity at pH4.0-5.0, and then the enzyme activity increased with increasing pH, and the enzyme reached the maximum value at pH8.0, from which it was found that the xanthine oxidase had the best stability in Tris-HCl buffer at pH 8.0.

2) Effect of temperature on XOD enzyme Activity and stability

In a buffer solution with the pH value of 7.5, enzyme activity at different temperatures is measured according to the method, and the optimum reaction temperature of XOD is determined. The enzyme solution is respectively kept at 30 ℃, 40 ℃, 50 ℃, 55 ℃, 60 ℃ and 70 ℃ for 30min, is rapidly cooled to room temperature, the residual enzyme activity is measured, and a temperature-enzyme activity curve is prepared, wherein the result is shown in figure 10, the enzyme activity change is not obvious between 30 ℃ and 50 ℃, 60 ℃ and 80 ℃, and the activity reaches the highest at 55 ℃.

And (3) keeping the enzyme solution at different temperatures for 30min, determining the change condition of the enzyme activity, and researching the thermal stability of the XOD. As a result, as shown in FIG. 11, the temperature greatly affected the stability of xanthine oxidase. The enzyme activity is basically not lost after 30min of treatment at the temperature of 30-50 ℃, and the enzyme basically loses the activity after 30min of treatment at the temperature of 60 ℃.

3) Determination of kinetic constants of XOD

Xanthine with different concentrations of 0.08, 0.16, 0.2, 0.32, 0.4mM, etcThe XOD enzyme activity of the substrate is measured, and the influence of the substrate on the enzymatic reaction speed is observed. According to the Lineweaver-Burk (double reciprocal mapping method) as shown in fig. 12, the linear equation is (1/V) ═ 0.5091 × (1/S) +7.6272, and the correlation coefficient R²The Michaelis constant Km of xanthine oxidase was 0.06675mM, and the maximum reaction rate was 0.1303mM ∙ min, to obtain 0.9958.

4) Effect of Metal ions on XOD enzymatic Activity

Different metal ions (final concentration of 2mM/L) were added to the enzyme solution, and the residual enzyme activity was measured after incubation at 25 ℃ for 2 hours. Relative enzyme activity was calculated using an enzyme solution without added metal ions as a control. The results are shown in Table 1, the metal ion Mg²⁺、Ca²⁺Has slight promoting effect on xanthine oxidase activity, Co²⁺、Mn²⁺、Ba²⁺Has certain inhibition effect on enzyme activity. And adding Ag into the reaction system⁺、Hg²⁺、 Pb²⁺、Zn²⁺、Cu²⁺、Fe²⁺、Fe³⁺No enzyme activity was detected after ionization and a significant precipitate was seen. The reason for analysis is that the reaction system is very insoluble in water, and the reaction system is made up of 0.025M/L NaOH to prepare a xanthine solution, so Ag⁺、Hg²⁺、Pb²⁺、Zn²⁺、Cu²⁺、Fe²⁺、Fe³⁺The plasma metal ions are all reacted with OH^-The reaction produced a precipitate.

TABLE 1 Effect of different Metal ions on enzyme Activity

Sequence listing

<110> Wuhan university of light industry

<120> xanthine oxidase capable of being used for clinical detection, encoding gene thereof and application thereof

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Claims

1. A recombinant strain for expressing xanthine oxidase is obtained by transforming an expression vector containing a xanthine oxidase expression cassette into an expression host, wherein the xanthine oxidase expression cassette consists of a promoter and a xanthine oxidase gene which is connected to the downstream of the promoter, the sequence of the xanthine oxidase gene is SEQ ID NO. 1 or a synonymous mutant thereof, and the expression host is recombinant engineering bacteria pAW 340.

2. A xanthine oxidase expressed by the recombinant strain according to claim 1.

3. Use of the xanthine oxidase of claim 2 for the preparation of a reagent for detecting the concentration of xanthine and/or hypoxanthine.

4. A method of oxidizing xanthine and/or hypoxanthine in a solution, comprising the step of adding to said solution the xanthine oxidase of claim 2.

5. The method of claim 4, wherein the temperature of the solution is 25-55 ℃ and the pH of the solution is 6.0-9.0.

6. The method of claim 4 or 5, wherein the solution is free of metal ions or comprises Mg²⁺、Ca²⁺、Mo⁶⁺、Co²⁺Or Mn²⁺。