阅读说明：本技术 测定酶活性的方法 (Method for measuring enzyme activity ) 是由 马龙华 李宁 顾好粮 李晓雯 高利艳 沈鹏飞 王晓东 孙斌 王利 刘松坡 谢文君 于 2019-10-10 设计创作，主要内容包括：本发明适用于酶检测技术领域,提供了一种测定酶活性的方法,包括以下步骤：S1：对金属蛋白酶的酶切底物进行重组蛋白纯化；S2：对酶彻底物进行酶切反应；S3：读取酶切反应后的金属靶板,并对样本内的酶切活性进行计算。借此,本发明灵敏度、准确性好,操作简便,价格便宜,应用范围广泛。(The invention is applicable to the technical field of enzyme detection, and provides a method for measuring enzyme activity, which comprises the following steps: s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease; s2: carrying out enzyme digestion reaction on the enzyme-treated product; s3: and reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample. Therefore, the method has the advantages of good sensitivity and accuracy, simple and convenient operation, low price and wide application range.)

1. A method for measuring enzyme activity, comprising the steps of:

s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease;

inserting the amino acid fragment and histidine at the tail end of the amino acid fragment into a bacterial expression vector and cloning the amino acid fragment and the histidine, then collecting the bacteria, cracking the bacteria, performing centrifugal treatment after cracking, collecting supernatant of the bacteria after centrifugal treatment, then adding buffer solution with the same volume for dilution, and eluting the diluted liquid by using a reducing ion column to further obtain a required enzyme substrate;

s2: carrying out enzyme digestion reaction on the enzyme-treated product;

diluting an enzyme substrate by using a reaction buffer solution, mixing a serum standard substance with a substrate solution obtained by diluting a measured serum sample, incubating the mixed liquid, heating the incubated solution to a preset temperature, and stopping the reaction;

s3: reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample;

adding an activation buffer solution into a metal target plate which is required and enriched with enzyme digestion products, oscillating and incubating, washing the metal target plate, adding a binding buffer solution, oscillating and incubating for the first time, absorbing residual solution on the target plate, adding the sample obtained in the step S2 into the target plate, oscillating, incubating and washing again, taking out the target plate, absorbing the residual solution, adding a substrate, reading the target plate by using an instrument, and calculating enzyme digestion activity by using the read peak area.

2. The method for measuring the enzymatic activity according to claim 1, wherein the amino acid fragment is D1596 to R1668 which are 73 amino acid fragments of ristomycin cofactor, and the histidine is 6 histidines at the terminal of ristomycin cofactor.

3. The method for measuring enzymatic activity of claim 2, wherein the sense strand and the antisense strand of the ristomycin cofactor are 5-cgggatccGAGGCACAGTCCAAAGGGGACA-3 and 5-cggaattc, respectivelyTCAGTGATG GTGATGGTGATGTCGGGGGAGCGTCTCAAAGTCC-3_。

4. The method for measuring enzyme activity according to claim 1, wherein the bacterial expression vector is pGEX6p-1 with GST protein at N-terminal, and after cloning of the bacterial expression vector is completed, BL21 expressing bacteria are transfected.

5. The method for measuring enzyme activity according to claim 1, wherein the lysis is ultrasonic lysis, the buffer diluent is PBS buffer solution with the same volume as the supernatant, and the reducing ion column is a reducing nickel ion column.

6. The method for measuring enzyme activity according to claim 1, wherein the dilution of enzyme concentrate in S2 is 0.1ug/ul, the reaction buffer comprises 5mM sodium chloride and 1mM barium chloride, and the pH is 7.5.

7. The method for measuring enzyme activity according to claim 1, wherein the incubation time in S2 is 1h, and the incubation is performed in a water bath at 37 ℃ and the preset temperature in S2 is 95 ℃.

8. The method of claim 1, wherein the activation buffer comprises 100mM copper sulfate, the binding buffer comprises an elution buffer and an equilibration buffer, and the elution buffer is 1XPBS, the equilibration buffer is 1 mh epes and has a PH of 7.0, and the substrate is sinapic acid.

9. The method for measuring the enzymatic activity according to claim 1, wherein the first shaking incubation is two shaking incubations with 5min, the second shaking incubation time is 30min, and the second shaking incubation is followed by elution with elution buffer for 3 times, 5min each time, and finally two washes with water.

10. The method of claim 9, wherein the amount of the substrate added is 1ul, and the substrate is added once after air-drying after the addition, and the method further comprises the step of adding the substrate once again after the air-dryingThe model of the instrument is Ebio Reader^TM3700 analyzing the target peak and internal reference peak area by software in the instrument, dividing the target peak by the internal reference peak area to obtain X-axis, and calculating the enzyme digestion activity according to the peak area of the sample by making a first-order curve with the standard serum concentration as Y-axis.

Technical Field

The invention relates to the technical field of enzyme detection, in particular to a method for measuring enzyme activity.

Background

Thrombotic microangiopathy mainly includes four types of diseases: thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, malignant hypertensive renal damage, preeclampsia renal damage.

The thrombotic microangiopathy is clinically mainly characterized by thrombocytopenia, hemolytic anemia and organ involvement caused by platelet thrombosis in microcirculation, and the clinical manifestations are related to the pathological change range of the thrombotic microangiopathy and functional disorders caused by involvement of different organs.

The clinical departments involved in the disease are very wide, patients can often see a doctor in different departments such as nephrology department, hematology department, neurology department, obstetrics and gynecology department, dermatology department, cardiovascular department, respiratory department and the like, and if a doctor who receives a doctor lacks understanding of the disease, serious missed diagnosis is often caused, so that the improvement of the cognition degree of the disease becomes a focus of attention in relevant professional fields at home and abroad at present.

The clinical manifestations of thrombotic microangiopathy are very similar, but the disease mechanism is completely different, wherein the biggest difference of thrombotic thrombocytopenic purpura is that the enzymatic activity of metalloprotease has significant difference. The activity of metalloprotease in patients with thrombotic thrombocytopenic purpura, whether hereditary or acquired, is generally less than 5%, while the activity of metalloprotease is lost in the onset of other types of thrombotic thrombocytopenic purpura, so that the activity of metalloprotease in patients with other types of thrombotic thrombocytopenic purpura is normal. .

Early detection technologies include an immunoradiometric assay, a collagen binding method and ristomycin cofactors, wherein a substrate is a full-fragment ristomycin cofactor, a polymer is required to be unfolded by adding a denaturant, but the denaturant is not contained in a human body, physiological conditions are not met, the reaction time is long, and about 1-2 days often cause that a patient loses life because of the fact that the patient cannot be diagnosed and treated in time.

Fluorescence resonance energy transfer method: the substrate FRETS-vWF73 is chemically synthesized, and the fluorescence quenching effect can be reduced after the substrate is specifically cleaved, namely, the fluorescence can be enhanced after the normal human plasma and the substrate act, and the influence of patients with the activity deficiency of the metalloprotease is avoided or the fluorescence is reduced. The technology needs a laboratory to prepare a multifunctional microplate reader with a high price, has a fluorescence detection function, and has high cost of chemically synthesized substrates, so that most patients abandon detection due to incapability of burden.

The enzyme activity of the metalloprotease is measured by enzyme-linked immunosorbent assay, which is a popular method in recent years, based on the double-antibody sandwich method technology, an enzyme-labeled plate hole with a specific antibody to a substrate is pre-coated, the substrate and a plasma sample are sequentially added for mixed incubation, an enzyme-labeled antibody and a color developing solution are added, after the substrate and the plasma sample containing the metalloprotease are mixed for incubation, the substrate is cracked by the metalloprotease, and the absorbance value of an enzyme-labeled instrument is lower. The absorbance value is higher if the metalloprotease activity in the plasma sample is lower or a congenital metalloprotease defect is present. The recombinant protein has large molecular weight, complex testing technical process and long time consumption, is difficult to meet the requirement of screening large-scale thrombotic thrombocytopenic purpura, and is not suitable for the clinical requirement of emergency diagnosis and treatment of patients in an emergency room.

In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.

Disclosure of Invention

In view of the above-mentioned drawbacks, the present invention provides a method for measuring enzyme activity, which can rapidly and effectively detect enzyme activity, reduce treatment cost, and improve cure rate.

In order to achieve the above object, the present invention provides a method for measuring an enzymatic activity, comprising the steps of:

s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease;

inserting the amino acid fragment and histidine at the tail end of the amino acid fragment into a bacterial expression vector and cloning the amino acid fragment and the histidine, then collecting the bacteria, cracking the bacteria, performing centrifugal treatment after cracking, collecting supernatant of the bacteria after centrifugal treatment, then adding buffer solution with the same volume for dilution, and eluting the diluted liquid by using a reducing ion column to further obtain a required enzyme substrate;

s2: carrying out enzyme digestion reaction on the enzyme-treated product;

diluting an enzyme substrate by using a reaction buffer solution, mixing a serum standard substance with a substrate solution obtained by diluting a measured serum sample, incubating the mixed liquid, heating the incubated solution to a preset temperature, and stopping the reaction;

s3: reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample;

adding an activation buffer solution into a metal target plate which is required and enriched with enzyme digestion products, oscillating and incubating, washing the metal target plate, adding a binding buffer solution, oscillating and incubating for the first time, absorbing residual solution on the target plate, adding the sample obtained in the step S2 into the target plate, oscillating, incubating and washing again, taking out the target plate, absorbing the residual solution, adding a substrate, reading the target plate by using an instrument, and calculating enzyme digestion activity by using the read peak area.

According to the method for measuring the enzyme activity, the amino acid fragment is D1596-R1668 of 73 amino acid fragments in ristomycin cofactor, and the histidine is 6 histidine at the tail end of the ristomycin cofactor.

According to the method for measuring an enzymatic activity of the present invention, the sense strand and the antisense strand of the ristomycin cofactor are each5-cgggatccGAGGCACAGTCCAAAGGGGACA-3 and 5-cggaattcTCAGTGATGGTGATGGTGATGTCGGGGGAGCGTCTCAAAGTCC-3_。

According to the method for determining enzyme activity, the bacterial expression vector is pGEX6p-1 with GST protein at the N end, and after cloning of the bacterial expression vector is completed, BL21 expression bacteria are transfected.

According to the method for determining the enzymatic activity, ultrasonic lysis is adopted for lysis, the buffer diluent is diluted by PBS buffer solution with the volume equal to that of the supernatant, and the reducing ion column is a reducing nickel ion column.

According to the method for measuring an enzyme activity of the present invention, the dilution of the enzyme concentrate in S2 was 0.1ug/ul, the reaction buffer included 5mM sodium chloride and 1mM barium chloride, and the pH was 7.5.

According to the method for determining the enzyme activity, the incubation time in the S2 is 1h, and a water bath kettle is adopted for incubation, the temperature of the water bath kettle is 37 ℃, and the preset temperature in the S2 is 95 ℃.

According to the method for measuring enzyme activity of the present invention, the activation buffer comprises 100mM copper sulfate, the binding buffer comprises an elution buffer and an equilibration buffer, the elution buffer is 1XPBS, the equilibration buffer is 1mMHEPES and has a pH of 7.0, and the substrate is sinapic acid.

According to the method for determining the enzymatic activity, the first shaking incubation is 5min shaking incubation twice, the second shaking incubation time is 30min, and the elution buffer solution is adopted to elute for 3 times after the second shaking incubation, each time for 5min, and finally the water is washed twice.

According to the method for measuring the enzymatic activity, the addition amount of the substrate is 1ul, the substrate is added once after the addition is finished and air-dried, and the model of the device is an Ebio Reader^TM3700 analyzing the target peak and internal reference peak area by software in the instrument, dividing the target peak by the internal reference peak area to obtain X-axis, and calculating the enzyme digestion activity according to the peak area of the sample by making a first-order curve with the standard serum concentration as Y-axis.

The invention provides a method for measuring enzyme activity, which comprises the following steps:

s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease;

inserting the amino acid fragment and histidine at the tail end of the amino acid fragment into a bacterial expression vector and cloning the amino acid fragment and the histidine, then collecting the bacteria, cracking the bacteria, performing centrifugal treatment after cracking, collecting supernatant of the bacteria after centrifugal treatment, then adding buffer solution with the same volume for dilution, and eluting the diluted liquid by using a reducing ion column to further obtain a required enzyme substrate;

s2: carrying out enzyme digestion reaction on the enzyme-treated product;

diluting an enzyme substrate by using a reaction buffer solution, mixing a serum standard substance with a substrate solution obtained by diluting a measured serum sample, incubating the mixed liquid, heating the incubated solution to a preset temperature, and stopping the reaction;

s3: reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample;

adding an activation buffer solution into a metal target plate which is required and enriched with enzyme digestion products, oscillating and incubating, washing the metal target plate, adding a binding buffer solution, oscillating and incubating for the first time, absorbing residual solution on the target plate, adding the sample obtained in the step S2 into the target plate, oscillating, incubating and washing again, taking out the target plate, absorbing the residual solution, adding a substrate, reading the target plate by using an instrument, and calculating enzyme digestion activity by using the read peak area.

The invention has the beneficial effects that:

1. the detection method is rapid and effective, and the detection is accurate.

2. All suspected cases of thrombotic microangiopathy are examined and all cases with metalloprotease activity less than 5% are selected and these patients will be treated as thrombotic thrombocytopenic purpura. For more than 5% of cases with enzyme activity, further diagnosis will be carried out and completely different treatments will be started.

3. For all cases of thrombotic thrombocytopenic purpura, the enzyme activity of the metalloprotease needs to be regularly detected during the treatment process and in the long-term monitoring after discharge, thereby better treating the patients correctly, reducing the treatment cost and effectively improving the survival rate and the life quality of the patients.

Drawings

FIG. 1 is a schematic diagram of the apparatus of the present invention;

FIG. 2 is a graph showing the values of enzyme activity read by the apparatus of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and embodiments, it being understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

Referring to fig. 1, the present invention provides a method for measuring an enzymatic activity, comprising the steps of:

s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease;

inserting the amino acid fragment and histidine at the tail end of the amino acid fragment into a bacterial expression vector and cloning the amino acid fragment and the histidine, then collecting the bacteria, cracking the bacteria, performing centrifugal treatment after cracking, collecting supernatant of the bacteria after centrifugal treatment, then adding buffer solution with the same volume for dilution, and eluting the diluted liquid by using a reducing ion column to further obtain a required enzyme substrate;

s2: carrying out enzyme digestion reaction on the enzyme-treated product;

diluting an enzyme substrate by using a reaction buffer solution, mixing a serum standard substance with a substrate solution obtained by diluting a measured serum sample, incubating the mixed liquid, heating the incubated solution to a preset temperature, and stopping the reaction;

s3: reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample;

adding an activation buffer solution into a metal target plate which is required and enriched with enzyme digestion products, oscillating and incubating, washing the metal target plate, adding a binding buffer solution, oscillating and incubating for the first time, absorbing residual solution on the target plate, adding the sample obtained in the step S2 into the target plate, oscillating, incubating and washing again, taking out the target plate, absorbing the residual solution, adding a substrate, reading the target plate by using an instrument, and calculating enzyme digestion activity by using the read peak area.

Preferably, the amino acid fragment of the invention is D1596-R1668 which is a 73 amino acid fragment of ristomycin cofactor, and the histidine is 6 histidines at the end of the ristomycin cofactor.

In addition, the sense strand and the antisense strand of the ristomycin cofactor of the present invention are 5-cgggatccGAGGCACAGTCCAAAGGGGACA-3-and 5-cggaattc, respectivelyTCAGTGATGGTGATGGTGATGTCGGGGGAGCGTCTCAAAGTCC-3_。

Further, the bacterial expression vector is pGEX6p-1 with GST protein at the N end, and after cloning of the bacterial expression vector is completed, BL21 expression bacteria are transfected.

Preferably, the cracking is performed by ultrasonic cracking, the buffer diluent is diluted by PBS buffer solution with the same volume as the supernatant, and the reducing ion column is a reducing nickel ion column.

Ebio Reader^TM3700 the system operating steps can be divided into the following steps:

1. directly spotting the sample to be analyzed on a target plate array for reaction, and specifically binding protein polypeptide in the sample to the target plate for reaction;

2. washing the target plate to wash away the small molecular substances which are not combined and are not specifically combined;

3. adding a matrix on the target plate, and converting the energy of laser into heat energy when the target plate is irradiated by the laser so as to ionize a sample;

4. after absorbing energy, protein polypeptide in the sample is desorbed and ionized, and flies to an ion detector through a vacuum tube, and different protein polypeptides are separated according to different mass-to-charge ratios because ions with different mass-to-charge ratios fly in the flight tube for different time. The mass-to-charge ratio (m/z) of ions detected, i.e. determined, from the differences in the time of flight of the arriving detector is proportional to the time of flight (t) of the ions, and the formula is:

in the formula:

m is the mass number of ions;

z is the charge carried by the ion;

e-elementary charge;

u-accelerating field voltage, V;

l-flight duct length, m;

t-time of flight, s.

As shown in fig. 1, Ebio Reader^TM3700 the instrument is composed of the host computer, computer and printer, the host computer is composed of laser, target plate transmission mechanism, ion source, flight tube, detector, signal line, data acquisition card, vacuum pump group, etc.

The metalloprotease (ADAMTS13) contains 1427 amino acid residues, including a hydrophobic signal peptide, a propeptide, a metalloprotease domain, a disintegrin domain, a thrombospondin repeat motif domain (TSP1), a cysteine-rich domain, a spacer and two CUB domains.

Research shows that a D1596-R1668 functional sequence consisting of 73 amino acid residues in an amino acid sequence of a ristocetin cofactor (vWF) functional region can be effectively cut by ADAMTS 13.

Obtaining of ristocetin cofactor (vWF73) DNA fragment: the DNA fragment of vWF73 was amplified by PCR using human umbilical vein endothelial cells as plasmid templates. The primer sequences were designed as follows:

sense strand:

5_-cgggatccGAGGCACAGTCCAAAGGGGACA-3_，

antisense strand:

5_-cggaattcTCAGTGATGGTGATGGTGATGTCGGGGGAGCGTCTCAAAGTCC-3_.

the two primers introduce BamHI and Hindm cleavage sites (lower case English letter portions), respectively, and insert a 6XHis Tag (underlined portion) at each C-terminus.

The method comprises the following specific steps: (1) the ADAMTS13 enzyme digestion substrate GST-vWF73-6XHis recombinant protein is expressed and purified; (2) carrying out enzyme digestion reaction; (3) the enzyme digestion product of ADAMTS13 is enriched by a metal target plate and then is subjected to Ebio Reader^TM3700 the assay is identified and the enzymatic activity of ADAMTS13 in the sample is calculated by the analysis software. The data obtained were analyzed computationally to quantitatively derive an accurate value for calculating the activity of ADAMTS13 enzyme, as shown in FIG. 2.

Step S3 includes a kit, which includes a substrate, an internal standard, a diluent, a buffer, a binding solution, an eluent, and a matrix.

The components are as follows:

(1) deionized water

(2) Reaction buffer (5mM sodium chloride, pH 7.5,1mM barium chloride)

(3) Activation buffer (100mM copper sulfate)

(4) Elution buffer (1XPBS)

(5) Equilibration buffer (1mM HEPES, pH 7.0)

(6) Substrate (erucic acid)

(7) Substrate (GST-vWF73-6XHis recombinant protein)

The invention has the following advantages:

1. the detection method is rapid and effective, and the detection is accurate.

2. All suspected cases of thrombotic microangiopathy were examined and all cases with ADAMTS13 enzyme activity less than 5% were selected and these patients were treated as Thrombotic Thrombocytopenic Purpura (TTP). For more than 5% of cases with enzyme activity, further diagnosis will be carried out and completely different treatments will be started.

3. For all TTP cases, the activity of ADAMTS13 enzyme needs to be regularly detected during the treatment process and long-term monitoring after discharge, so that the correct treatment is better performed on patients, the treatment cost is reduced, and the survival rate and the life quality of the patients are effectively improved.

In summary, the present invention provides a method for determining enzyme activity, comprising the steps of:

s1: purifying recombinant protein of an enzyme digestion substrate of the metalloprotease;

inserting the amino acid fragment and histidine at the tail end of the amino acid fragment into a bacterial expression vector and cloning the amino acid fragment and the histidine, then collecting the bacteria, cracking the bacteria, performing centrifugal treatment after cracking, collecting supernatant of the bacteria after centrifugal treatment, then adding buffer solution with the same volume for dilution, and eluting the diluted liquid by using a reducing ion column to further obtain a required enzyme substrate;

s2: carrying out enzyme digestion reaction on the enzyme-treated product;

diluting an enzyme substrate by using a reaction buffer solution, mixing a serum standard substance with a substrate solution obtained by diluting a measured serum sample, incubating the mixed liquid, heating the incubated solution to a preset temperature, and stopping the reaction;

s3: reading the metal target plate after the enzyme digestion reaction, and calculating the enzyme digestion activity in the sample;

adding an activation buffer solution into a metal target plate which is required and enriched with enzyme digestion products, oscillating and incubating, washing the metal target plate, adding a binding buffer solution, oscillating and incubating for the first time, absorbing residual solution on the target plate, adding the sample obtained in the step S2 into the target plate, oscillating, incubating and washing again, taking out the target plate, absorbing the residual solution, adding a substrate, reading the target plate by using an instrument, and calculating enzyme digestion activity by using the read peak area.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.