CN111487206B

CN111487206B - Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent

Info

Publication number: CN111487206B
Application number: CN202010000321.7A
Authority: CN
Inventors: 张启飞; 金石; 龚俊; 王贵利; 封建新; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2020-01-02
Publication date: 2023-11-10
Anticipated expiration: 2040-01-02
Also published as: CN117054643A; CN116718764A; CN116124721A; CN111650135A; CN111537451A; CN112285037A; CN116338215A; CN116840467A; CN111504920A; CN116718761A; CN116626281A; CN116735512A; CN116148198A; CN117030640A; CN112285038A; CN111537452B; CN116359146A; CN116699122A; CN116773795A; CN111487207A

Abstract

The application relates to a glucose-6-phosphate dehydrogenase mutant and application thereof in preparing a vancomycin detection reagent. Specifically, the mutant glucose 6-phosphate dehydrogenase of the present application comprises one mutation or a combination thereof selected from the group consisting of: d306C, D375C, G426C. The detection kit prepared by using the glucose 6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time, accurate quantification and suitability for high-throughput detection.

Description

Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent

Technical Field

The application relates to the field of biological detection, in particular to mutant enzyme glucose 6-phosphate dehydrogenase (G6 PDH for short) and application thereof in a vancomycin detection kit.

Background

Hapten, some small molecule substances (molecular weight less than 4000 Da) alone are not able to induce an immune response, i.e. are not immunogenic, but are immunogenic when crosslinked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine, inducing an immune response. These small molecule substances can bind to the response effect products, are antigenic, are only immunoreactive, are not immunogenic, and are also called incomplete antigens.

Hapten can bind to the corresponding antibody to generate antigen-antibody reaction, and antigen which can not independently excite human or animal body to generate antibody can not be generated. It is only immunoreactive, not immunogenic, also known as incomplete antigen. Most polysaccharides, lipids, hormones and small molecule drugs belong to the hapten group. If hapten is chemically bound to a protein molecule (carrier), new immunogenicity is obtained and the animal is stimulated to produce the corresponding antibody. Hapten, once bound to a protein, constitutes an antigenic cluster of the protein. Some chemically active substances (such as penicillin, sulfonamides, etc.) which have a smaller molecular weight than the general hapten but a specific structure are called simple haptens.

Small molecule antigens or haptens, which lack two or more sites available for sandwich methods, cannot be assayed by the double antibody sandwich method, and are often in competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with the solid phase antibody. The more the antigen content in the specimen, the less the enzyme-labeled antigen is bound on the solid phase, and the lighter the color development. ELISA assay for small molecule hormones, drugs and the like is commonly used.

Vancomycin (Vancomycin) has the structural formula shown in the following figure:

vancomycin has a molecular weight of 1485.71, is a narrow-spectrum antibiotic, is effective only on gram-positive bacteria (such as streptococcus hemolyticus, pneumococcus and enterococcus), and is particularly sensitive to drug-resistant staphylococcus aureus.

The mechanism of action of vancomycin is to inhibit bacterial cell wall synthesis, which binds primarily to bacterial cell walls, and prevents certain amino acids from entering the cell wall glycopeptides. The medicine is clinically used for treating serious infection caused by penicillin-resistant staphylococcus aureus, such as pneumonia, endocarditis, septicemia and the like, and has better curative effects on infection caused by hemolytic streptococcus, septicemia and the like. Vancomycin can also be used to treat colitis and intestinal inflammation; vancomycin is also often used to prevent infection when devices such as cardiac catheters, intravenous catheters, and the like are installed.

Vancomycin can be used alone or in combination. Vancomycin has no cross resistance with other antibiotics, and very few resistant strains. Is mainly used for endocarditis, septicemia, pseudomembranous enteritis and the like.

Vancomycin is not absorbed by oral administration, 400mg is infused by single dose intravenous drip, the peak concentration of the blood is 25.18mg/L after infusion, the average blood concentration is 1.90mg/L in 8 hours, and the effective blood concentration can be maintained for 6 to 8 hours. A single intravenous drip of 800mg was performed, and the peak blood concentration was 50.07mg/L on average. After intravenous drip, the medicine is mainly excreted through the kidney, 400mg is dropped in a single intravenous drip, and the average total excretion rate in urine is 81.1% in 24 hours; the average total excretion rate in urine per intravenous drip 800mg in 24 hours was 85.9%.

The currently known vancomycin detection methods mainly comprise: high Performance Liquid Chromatography (HPLC), luminescence immunity, enzyme-linked immunosorbent assay (ELISA), etc. The HPLC method requires complex sample pretreatment, and has the advantages of long operation period and high cost; the luminous immunoassay method has the disadvantages of high reagent cost, inapplicability to detection of conventional therapeutic drugs and inapplicability to large-scale popularization.

The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to complex preparation process and large batch-to-batch difference.

The prior art CN108717117a describes a vancomycin detection kit. However, the prior art methods rely on activation of the reactive groups carried by the small molecule drug (vancomycin) itself, followed by reaction with the enzyme. Such coupling methods can occur when multiple vancomycin are linked to the same glucose hexaphosphate dehydrogenase, and it is difficult to ensure consistency of the coupling sites, and to ensure orientation between the small molecule drug and the enzyme 1:1, resulting in large batch-to-batch variation.

Disclosure of Invention

In view of the needs in the art, the application provides a novel glucose-6-phosphate dehydrogenase mutant and application thereof in preparing a vancomycin detection kit.

According to some embodiments, a glucose 6-phosphate dehydrogenase mutant is provided. Unlike the mutants of glucose 6 phosphate dehydrogenase of the prior published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose 6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: d306C, G426C, D375C.

According to some embodiments, there is provided a glucose 6-phosphate dehydrogenase mutant, the glucose 6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

According to some embodiments, a polynucleotide encoding a glucose 6-phosphate dehydrogenase mutant of the present application is provided.

According to some embodiments, there is provided an expression vector comprising a polynucleotide of the application.

According to some embodiments, there is provided a host cell comprising an expression vector of the application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate which is a glucose 6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: and n is coupled.

In some embodiments, n is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

In some specific embodiments, the molar ratio of glucose 6-phosphate dehydrogenase mutants of the present application to hapten is preferably 1:1.

in some specific embodiments, the hapten has a molecular weight of 100Da to 4000Da, for example: 100. 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

According to the present application, the skilled person will understand that "hapten" also includes the form of its derivative. In order to facilitate the coupling with glucose-6-phosphate dehydrogenase, haptens (e.g., vancomycin) that do not themselves carry a coupling group (e.g., a group that reacts with a thiol group) may be engineered to carry a linker for covalent binding to the thiol group. Thus, in the present application, hapten derivatives refer to haptens engineered to bear a thiol-reactive group.

The hapten is selected from the group consisting of: small molecule drugs (e.g., antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

Hapten such as, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including digitoxin), zymophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, uronidine metabolite cotinine, uromorphine, uromonophenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactation, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, anti-triiodothyronine, free thyroxine, free triiodothyronine, cortisol urine 17-hydroxycortic steroids, urine 17-ketosterols, dehydroepiandrosterone and sulfates, aldosterone, uronolamine mandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 alpha-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C peptide, gastrin, plasma prostaglandin, plasma 6-keto prostaglandin f1α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, natriuretic acid adenosine cyclophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a specific embodiment, the hapten is vancomycin or a derivative thereof.

In a specific embodiment, the hapten is a vancomycin derivative bearing a sulfhydryl reactive group such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

In a specific embodiment, the hapten is a vancomycin derivative, as shown in formula I:

in some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

According to some embodiments, there is provided an agent comprising a conjugate of the application.

According to some embodiments, there is provided the use of a glucose 6-phosphate dehydrogenase mutant of the application in the preparation of a vancomycin detection reagent.

According to some embodiments, there is provided the use of a conjugate of the application in the preparation of a vancomycin detection reagent.

In specific embodiments, the detection reagent is selected from the group consisting of: ELISA detection reagent, chemiluminescent detection reagent, homogeneous ELISA detection reagent and latex enhanced turbidimetry detection reagent.

In a specific embodiment, the detection reagent is preferably a reagent for competition-based detection.

According to some embodiments, there is provided the use of a conjugate of the application in the preparation of a vancomycin detection device.

In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., 96-well plate), such as a plate coated with reagents according to the application.

In a specific embodiment, the detection device may be prepared in the form of particles (e.g. latex, magnetic beads), such as particles coated with the reagent according to the application.

According to some embodiments, there is provided a vancomycin detection kit comprising:

-a first reagent comprising a substrate, a buffer and a vancomycin antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the application and a buffer;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0 μg/ml to 100 μg/ml vancomycin; and

-optionally, a quality control comprising 10mM to 500mM buffer, 4 μg/ml to 45 μg/ml vancomycin.

According to one embodiment, there is provided a vancomycin detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer,

5mM to 50mM substrate,

0.5 to 5 (preferably 1 to 3) mg/L vancomycin antibody,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer,

10 to 500 (preferably 50 to 200) ng/mL of a conjugate according to the application,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative.

In some embodiments, the buffer is selected from one or a combination of the following: TAPS, tromethamine buffer, phosphate buffer, tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, and trimethylol methane buffer; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 50 to 100mM; the pH of the buffer is 7 to 8.

In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; bovine serum albumin is preferred.

In some embodiments, the surfactant is selected from one or a combination of the following: brij35, triton X-100, triton X-405, tween20, tween30, tween80, coconut fatty acid diethanolamide, AEO7, preferably Tween20.

In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, biological preservative PC (such as PC-300), merthiolate; the azide is selected from: sodium azide and lithium azide.

In some embodiments, the substrate comprises: glucose-6-phosphate, beta-nicotinamide adenine dinucleotide.

In some specific embodiments, the vancomycin antibody is derived from: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans.

In some specific embodiments, the vancomycin antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen binding fragments.

According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:

1) Providing a vancomycin derivative according to the application, in particular in an aprotic solvent (such as, but not limited to, acetonitrile, dimethylformamide, dimethylsulfoxide);

2) Providing a glucose 6-phosphate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment such as, but not limited to PBS, tris, TAPS, TAPSO, said buffer having a pH of 6.0 to 8.0);

3) At 18 ℃ to 28 ℃, the vancomycin derivative and the glucose 6-phosphate dehydrogenase mutant are mixed according to a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow coupling of the vancomycin derivative and the glucose 6-phosphate dehydrogenase mutant to occur, to give the seed conjugate;

4) The seed conjugate is optionally purified, e.g., desalted, etc., as desired.

In some embodiments, the contacting molar ratio of enzyme to hapten in the reaction system is 1: n, where n is 1 to 500, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the foregoing values thereof.

In some specific embodiments, steps 1) and 2) may be interchanged or in parallel.

In some specific embodiments, the glucose-6-phosphate dehydrogenase comprises one or more free sulfhydryl groups prior to coupling, thereby allowing for a directed reaction with vancomycin.

Wild-type glucose 6-phosphate dehydrogenase does not contain a free thiol group, and thus in some specific embodiments, the glucose 6-phosphate dehydrogenase is genetically engineered to have an amino acid mutation at a particular site (306, 375, or 426) to a cysteine, thereby carrying a free thiol group.

Drawings

FIG. 1 is a vancomycin structural diagram.

FIG. 2 is a block diagram of vancomycin derivatives.

FIG. 3A. G6PDH (wild type) amino acid sequence (SEQ ID No. 1); is derived from Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides.

FIG. 3B.G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3C.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 3D.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

EXAMPLE 1 Synthesis of vancomycin derivatives

1. Synthesis of Compound 2

To a round bottom flask was added DCM, to which was added Compound 1 (100 mg,0.47 mmol) and DMF (2 drops) and oxalyl chloride (90 mg,0.74 mmol) was slowly added thereto under ice-bath, air-tight conditions, the reaction was heated under reflux for 2-4h, the solvent was removed under reduced pressure, and the excess oxalyl chloride was carried over with DCM (2-3 times) and used directly in the next step without purification.

2. Synthesis of vancomycin derivatives

Vancomycin (100 mg) was dissolved in DMF, then compound 2 synthesized in the above step was added to the reaction system, stirred at room temperature (18-28 ℃ C.) for about 2 hours, and HPLC-purified to give a vancomycin derivative (50 mg, 47%):

the structure of the product was confirmed by a conventional method.

This example allows vancomycin to carry a group that can bind to an enzyme.

EXAMPLE 2 coupling of vancomycin derivatives with G6PDH molecules

1. The coupling method of the application

The G6 PDH-vancomycin conjugate according to the application is coupled as follows: thiol-reactive groups (such as but not limited to maleimide groups) on the vancomycin derivative molecule are covalently bound to thiol groups on the G6PDH molecule.

1. The vancomycin derivative prepared in example 1 was dissolved in N, N-dimethylformamide (10 mg/ml);

g6pdh solution: g6pdh (mutant of the application or mutant of the prior art) was dissolved in PB 100mmol, naCl 100mmol, ph=8.0;

3. 200 mu l G of 6PDH solution was added to 750. Mu.l buffer (0.05M Na ₂ HPO ₄ 、150mM NaCl、10mM EDTA、0.1％ NaN ₃ Ph=7.2); then 50. Mu.l of an N, N-dimethylformamide solution of a vancomycin derivative was added thereto;

4. the mixed solution is fully vibrated for 2-3 hours at room temperature (18-28 ℃), desalted and protein peaks are collected, and the obtained product is the G6 PDH-vancomycin conjugate.

2. Control coupling method (refer to CN 108717117A)

Dissolving 5-30mg vancomycin in 100-500 mu L dimethylformamide, and slightly oscillating to dissolve; meanwhile, 10-40mg of 100-300KU G6PDH is dissolved in PBS buffer solution for shaking and uniform dissolution.

The vancomycin solution is slowly added into the G6PDH solution under stirring, meanwhile, 10-50 mu L of glutaraldehyde solution is slowly dripped, and after the dripping is finished, the solution is placed at 4 ℃ to be stirred and reacted overnight.

Purifying by G-25 gel chromatography column to obtain G6 PDH-vancomycin conjugate, and storing at 2-8deg.C.

EXAMPLE 3 preparation of the kit

The following kit for detecting vancomycin was prepared, which comprises:

reagent R1 comprising:

50mM TAPS，pH 8.0

15mM glucose 6-phosphate

15mM beta-nicotinamide adenine dinucleotide

2.0mg/L vancomycin antibody (commercially available antibody)

100mM NaCl

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

50mM Tris buffer, pH 8.0

100ng/mL G6 PDH-vancomycin conjugate

100mM NaCl

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration material: 20mM HEPES buffer, 0 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100 μg/ml vancomycin (or added as needed);

quality control product: 20mM HEPES buffer, 4-8. Mu.g/ml, 15-20. Mu.g/ml, 35-45. Mu.g/ml vancomycin (or added as needed).

The reagent (optionally comprising a quality control product and a calibrator) is assembled into the vancomycin homogeneous enzyme immunoassay kit.

Test case

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6 PDH-vancomycin) and unlabeled antigen (vancomycin) compete for being combined with quantitative antibody (vancomycin antibody), when the more the antibody is combined with the unlabeled antigen, the more the activity of the enzyme-labeled antigen is released, the more the enzyme-catalyzed substrate NAD+ generates NADH, and the absorbance change of the NADH is detected at the wavelength of 340nm, so that the content of the vancomycin in the liquid can be deduced.

TABLE 1 full automatic Biochemical instrument parameters

Detection method	Rate method
		Sample size	2.0μl
Reagent R1	150μl
		Reagent R2	50μl
Reading point	31-34 points
		Detection wavelength (Main)	340
Detection wavelength (auxiliary)	405
		Curve fitting mode	Spline

Detection example 1. Precision and linearity experiments of the kit of the present application

1. Calibration experiment

Vancomycin calibrator was dissolved in buffer (0.9% NaCl, 0.1% NaN) ₃ ) 6 kinds of calibration materials with the total concentration are prepared, the working volume of the calibration solution is 2-10 mu l, and then 100-200 mu l of reagent R1 and 50-100 mu l of reagent R2 are added. And detecting the main wavelength of 340nm and the auxiliary wavelength of 405nm by adopting a rate method, reading the absorbance change rate, and drawing a calibration curve. The establishment of the calibration curve in the present application is completed on Hitachi 7180, but other mainstream models (AU 680, attapulgite C16000, etc.) are available through detection.

2. Precision experiments

And (3) measuring high, medium and low quality control products and clinical samples by using the calibration curve established above, and detecting 20 times respectively. As shown in table 2, the sample test was repeated 20 times with a deviation of less than 3%.

TABLE 2 precision (for D306C mutant)

3. Linear experiments

Selecting a low-value sample and a high-value sample to dilute according to an arithmetic dilution method, repeatedly detecting each sample for 3 times, wherein the linear data is shown in table 3, and the linear formula is as follows:

y＝8.8026x+2.9902

R ^z ＝0.9985

TABLE 3 vancomycin kit (for D306C mutant) Linear data

Sample of	Measurement value 1	Measurement value 2	Measurement value 3	Mean value of	Theoretical value	Relative deviation of	Absolute deviation of
								0	1.99	1.99	2.23	2.11	1.84		0.27
1	11.80	10.50	12.50	11.50	11.64	-1.2％
								2	21.93	21.93	21.04	21.49	21.44	0.2％
3	32.00	31.50	31.80	31.65	31.24	1.3％
								4	41.50	40.21	41.30	40.76	41.03	-0.7％
5	50.10	51.60	50.90	51.25	50.83	0.8％
								6	58.90	59.75	57.86	58.81	60.63	-3.0％
7	69.88	71.21	70.85	71.03	70.42	0.9％
								8	80.11	80.34	79.88	80.11	80.22	-0.1％
9	89.00	90.11	91.12	90.62	90.02	0.7％
								10	98.31	96.55	101.00	98.78	99.81	-1.0％

Test example 2 anti-interference action on common drugs

30 compounds or drugs are selected as interferents, and when the vancomycin concentration is about 25 mug/ml and the interferent concentration is 500 mug/ml, the following interferents have no obvious interference (aiming at D306C mutant):

acetaminophen, phenacetin, cefazolin, furosemide, benflumethizine, kanamycin B, terramycin, hydrochlorothiazide, sisomicin, amikacin, prednisolone, cefalexin, fusidic acid, caffeine, lincomycin, penicillin, ibuprofen, spectinomycin, amphotericin B, prednisone, carbamazepine, gentamicin, cefamane, phenytoin, amikacin, kanamycin A, heparin, salicylic acid, clodronic acid.

Test example 3 on-board stability

The quality control products with high, medium and low concentrations are selected and detected three times every day or every other day, and the data show that the vancomycin detection kit (aiming at 375 mutant) provided by the application can be stable for more than two weeks in a calibration period. After the reagent was unsealed, it was set on a machine, and the test showed that the machine stability exceeded 40 days (table 6).

Detection example 4. Batch to batch differences for vancomycin detection kit

Three batches of the reagent (D306C mutant) of the application and the reagent prepared by the control coupling method were used for calibration respectively, and the absorbance variation difference of different batches was calculated.

TABLE 4 batch to batch differences

Detection example 5 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-vancomycin conjugate, the activity of the G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an added antibody and an experimental group without the added antibody is compared by detecting the change of the NADH amount, and the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. Reaction system

TABLE 5 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-vancomycin conjugate when the antibody is added with the antibody is not added with the antibody, and obtaining the inhibition condition of the antibody on the G6 PDH.

Antibody inhibition = change in absorbance of G6 PDH-vancomycin with antibody/change in absorbance of G6 PDH-vancomycin without antibody) ×100%.

Compared with the published mutation site (A45C), the mutant of the application has obvious improvement on the antibody inhibition rate, can reach more than 35 percent (G426C: 35 percent; D375C:50 percent) and can reach 56 percent (D306C) at most. Whereas the inhibition rates of the previously published mutation sites (e.g.A45C, K55C) were 32% and 41%.

While not being limited to a particular theory, it may be explained in part as: in comparison with the G6PDH mutant (A45C, K C) in the prior art, the mutation site (i.e. the site for introducing free sulfhydryl) in the enzyme mutant is the coupling site with hapten (such as hormone, small molecule drug, etc.). When hapten is combined with hapten specific antibody at this position, the steric hindrance formed has the greatest effect on the activity of G6PDH enzyme, and after mutation is introduced, the steric folding of the molecule cannot be substantially influenced. Therefore, the position of this mutation site is very important, and it is necessary to combine the activity of the G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The mutant of the enzyme has obvious improvement on the inhibition rate of the antibody. After the conjugate of the enzyme mutant and vancomycin is prepared into a kit, the reagent has obvious performance improvement in the aspects of the batch-to-batch variation coefficient, linearity, specificity and the like.

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Sequence listing

<110> Beijing Jiuqiang biotechnology Co., ltd

<120> glucose-6-phosphate dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent

<130> 390272CG

<150> 201910017764.4

<151> 2019-01-09

<150> 201910423122.4

<151> 2019-05-21

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 486

<212> PRT

<213> Leuconostoc mesenteroides (Leuconostoc pseudomesenteroides)

<400> 1

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Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

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Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

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Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

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Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

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Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

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Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

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Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

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Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 2

<211> 486

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (306)..(306)

<223> G6PDH mutant, D at position 306 replaced with C compared to wild type

<400> 2

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Cys Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 3

<211> 486

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (375)..(375)

<223> G6PDH mutant, substitution of D at position 375 with C compared to wild type

<400> 3

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Cys Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 4

<211> 486

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (426)..(426)

<223> G6PDH mutant, substitution of G at position 426 with C compared to wild type

<400> 5

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Cys Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims

1. A conjugate which is a mutant glucose-6-phosphate dehydrogenase and a vancomycin derivative in a molar ratio of 1:1, coupling;

the glucose 6-phosphate dehydrogenase mutant comprises one mutation as compared to the wild-type glucose 6-phosphate dehydrogenase selected from the group consisting of: d306C, D375C;

the glucose 6-phosphate dehydrogenase mutant is shown by a sequence selected from the following: SEQ ID No.2, SEQ ID No.3;

the vancomycin derivative is represented by formula I:

wherein,

m is an integer of 1 to 10.

2. The conjugate of claim 1, m is an integer from 1 to 5.