CN111487206A

CN111487206A - 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent

Info

Publication number: CN111487206A
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: 2020-08-04
Anticipated expiration: 2040-01-02
Also published as: CN116859035A; CN116840462A; CN117030640A; CN116840468A; CN111487207B; CN111693473A; CN116148198A; CN116718761A; CN116626281A; CN116735512A; CN116124721A; CN111504921A; CN116773827A; CN112285038A; CN111487207A; CN116298330A; CN111650135A; CN111678874A; CN116699125A; CN116773795A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a vancomycin detection reagent. Specifically, the glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations 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 and accurate quantification, and is suitable for high-throughput detection.

Description

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

The present application claims priority from 201910017764.4 filed on day 1/9 in 2019 and 201910423122.4 "glucose-6-phosphate dehydrogenase mutant and its use in the preparation of test agents" filed on day 21/5 in 2019, which are incorporated herein by reference.

Technical Field

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

Background

Haptens, some small molecular substances (molecular weight less than 4000Da), alone cannot induce an immune response, i.e. are not immunogenic, but can acquire immunogenicity when crosslinked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysine, and induce an immune response. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.

The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and will stimulate the production of corresponding antibodies in animals. Haptens, once bound to a protein, constitute an antigenic cluster of the protein. Some chemical active group substances (such as penicillin, sulfanilamide, etc.) with molecular weight smaller than that of general hapten and specific structure are called simple hapten.

The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with solid-phase antibody, the more the content of the antigen in the specimen is, the less the enzyme-labeled antigen is bound on the solid phase, the lighter the color is, and the more the E L ISA is used for measuring small molecular hormone, medicine, etc.

The structural formula of Vancomycin (Vancomycin) is shown as follows:

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

The mechanism of action of vancomycin is to inhibit bacterial cell wall synthesis, which is primarily associated with the bacterial cell wall, leaving certain amino acids unable to enter the glycopeptides of the cell wall. The medicine is mainly used for treating serious infection caused by penicillium resistant staphylococcus aureus, such as pneumonia, endocarditis, septicemia and the like, and has better curative effect on infection, septicemia and the like caused by hemolytic streptococcus. Vancomycin can also be used to treat colitis and intestinal inflammation; vancomycin is also often used to prevent infection when installing cardiac catheters, venous catheters, and the like.

Vancomycin can be administered alone or in combination. Vancomycin has no cross resistance with other antibiotics, and has few drug-resistant strains. It is mainly used for treating endocarditis, septicemia, pseudomembranous enteritis, etc.

The vancomycin is not absorbed by oral administration, 400mg of single dose of intravenous drip reaches the blood drug peak concentration of 25.18 mg/L after the drip is finished, the average blood concentration in 8 hours is 1.90 mg/L, the effective blood concentration can be maintained for 6 to 8 hours, 800mg of single intravenous drip is used, the average peak blood concentration is 50.07 mg/L, after the intravenous drip, the vancomycin is mainly excreted by the kidney, 400mg of single intravenous drip is used, the average total excretion rate in 24 hours of urine is 81.1%, 800mg of single intravenous drip is used, and the average total excretion rate in 24 hours of urine is 85.9%.

The currently known vancomycin detection methods mainly comprise methods such as high performance liquid chromatography (HP L C), luminescence immunization, enzyme-linked immunosorbent assay (E L ISA) and the like, the HP L C method needs complex sample pretreatment, is complex in operation, long in period and high in cost, and the luminescence immunization method is high in reagent cost, is not suitable for conventional therapeutic drug detection and is not more beneficial to large-scale popularization.

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

The prior art CN108717117A describes a vancomycin detection kit. However, the prior art methods rely on the activation of reactive groups carried by the small molecule drug (vancomycin) itself, followed by reaction with an enzyme. Such a conjugation method may result in the situation where multiple vancomycin are linked to the same glucose-hexametaphosphate dehydrogenase, and it is difficult to ensure consistency of conjugation sites, orientation between small molecule drug and enzyme 1: 1, resulting in large batch-to-batch variation.

Disclosure of Invention

In view of the needs in the art, the present application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in the preparation of vancomycin detection kits.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the previously published mutant of glucose-6-phosphate dehydrogenase of patent US006090567A (halogenated immunological systems using mutant glucose-6-phosphate dehydrogenes), 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, there is provided a polynucleotide encoding a glucose-6-phosphate dehydrogenase mutant of the present application.

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

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

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

in some specific embodiments, the hapten has a molecular weight of from 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 comprises forms of its derivatives. To facilitate conjugation to 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) can be engineered to carry a linker for covalent binding to the thiol group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.

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

Haptens are, for example, but not limited to, vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxigenin), mycophenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites of urocortin, cotinine, morphine, derivatives of uropamine, neuropeptide tyrosine, plasma galangin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyroxine, cortisol, urinary 17-hydroxycorticosteroid, urinary 17-ketosteroid, epiandrosterone, testosterone, phytosterone, renin-B, renin-5, renin-B, renin-1, renin-renin, renin-angiotensin, testosterone, renin-angiotensin, renin-renin.

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

In particular embodiments, the hapten is a vancomycin derivative with a thiol reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

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

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

According to some embodiments, there is provided a reagent comprising a conjugate of the present application.

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

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

In specific embodiments, the detection reagent is selected from the group consisting of: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

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

According to some embodiments, there is provided the use of a conjugate of the present 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., a 96-well plate), such as a plate coated with a reagent according to the present application.

In particular embodiments, the detection device may be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with a reagent according to the present 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 present application and a buffer;

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

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

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

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

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

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

0.1 g/L to 5 g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

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

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

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

0.1 g/L to 5 g/L of preservative.

In some embodiments, the buffer is selected from one or a combination of TAPS, tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolmethane buffer, preferably phosphate buffer, at a concentration of 10 mmol/L to 500 mmol/L, preferably 50 to 100mM, and at a pH of 7 to 8.

In some embodiments, the stabilizing agent is selected from one or a combination of: 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: brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.

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

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

In some specific embodiments, the vancomycin antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, human.

In some specific embodiments, the vancomycin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, 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 present 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, buffer pH between 6.0 and 8.0);

3) (ii) mixing the vancomycin derivative and the glucose-6-phosphate dehydrogenase mutant at a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow the vancomycin derivative and the glucose-6-phosphate dehydrogenase mutant to be coupled to obtain the seed conjugate;

4) the conjugate is optionally subjected to purification, such as desalting treatment or the like, as required.

In some embodiments, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein 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.

In some specific embodiments, steps 1) and 2) are interchangeable or concurrent.

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

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

Drawings

FIG. 1 shows the structural diagram of vancomycin.

FIG. 2 shows the structural diagram of vancomycin derivative.

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

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(100mg, 0.47 mmol) and DMF (2 drops), to which was added oxalyl chloride (90mg, 0.74mmol) slowly with exclusion of air in an ice bath, the reaction was heated under reflux for 2-4h, the solvent was removed under reduced pressure, and the excess oxalyl chloride was taken up repeatedly (2-3 times) with DCM and used in the next step without purification.

2. Synthesis of vancomycin derivatives

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

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

This example allows vancomycin to have one group that can bind to an enzyme.

Example 2 coupling of vancomycin derivatives to G6PDH molecules

First, the coupling method of the present application

The coupling of the G6 PDH-vancomycin conjugate according to the present application was performed as follows: a thiol-reactive group (such as, but not limited to, a maleimide group) on a vancomycin derivative molecule is covalently bound to a thiol on a 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 present application or prior art mutant) was dissolved in PB 100mmol, NaCl100mmol, pH 8.0;

3. 200 μ l G6PDH solution was added to 750 μ l buffer solution (0.05M Na)₂HPO₄、 150mM NaCl、10mMEDTA、0.1％NaN₃pH 7.2); then 50. mu.l of an N, N-dimethylformamide solution of a vancomycin derivative was added thereto;

4. and (3) fully shaking the mixed solution at room temperature (18-28 ℃) for 2-3 hours, desalting, and collecting protein peaks to obtain a product, namely the G6 PDH-vancomycin conjugate.

Second, control coupling method (see method CN 108717117A)

5-30mg of vancomycin is dissolved in 100-500 mu L dimethylformamide and dissolved by slight shaking, and 10-40mg of 100-300KU G6PDH is dissolved in PBS buffer solution and dissolved uniformly by shaking.

Slowly adding the vancomycin solution into the G6PDH solution under stirring, slowly dropping 10-50 mu L glutaraldehyde solution, and after dropping, placing the solution at 4 ℃ for stirring reaction overnight.

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

Example 3 preparation of the kit

A kit for detecting vancomycin was prepared, which comprises:

reagent R1, comprising:

50mM TAPS，pH 8.0

15mM glucose 6-phosphate

15mM β -Nicotinamide adenine dinucleotide

2.0 mg/L vancomycin antibody (commercially available antibody)

100mM NaCl

1 g/L bovine serum albumin

1g/L Tween20

1 g/L sodium azide;

reagent R2, comprising:

50mM Tris buffer, pH 8.0

100ng/m L G6 PDH-vancomycin conjugate

100mM NaCl

1 g/L bovine serum albumin

1g/L Tween 20

1 g/L sodium azide;

calibration products: 20mM HEPES buffer, and 0. mu.g/ml, 5. mu.g/ml, 10. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml vancomycin (or added as needed);

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

And assembling the reagents (optionally containing quality control products and calibration products) into the vancomycin homogeneous enzyme immunoassay kit.

Example of detection

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, an enzyme-labeled antigen (such as G6 PDH-vancomycin) and a non-labeled antigen (vancomycin) compete for binding with a quantitative antibody (vancomycin antibody), when the antibody is more bound with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH + generated by an enzyme catalysis substrate NAD + is, and the absorbance change of NADH is detected at a wavelength of 340nm, so that the content of the vancomycin in the liquid can be calculated.

TABLE 1 parameters of fully automatic biochemical analyzer

Detection method	Velocity method
		Sample size	2.0μl
Reagent R1	150μl
		Reagent R2	50μl
Read point	Points 31-34
		Detecting wavelength (main)	340
Detecting wavelength (vice)	405
		Curve fitting method	Spline

Detection example 1 precision and linearity experiment of the kit of the present application

1. Calibration experiment

Vancomycin calibrator was dissolved in buffer (0.9% NaCl, 0.1% NaN)₃) 6 calibrators with different concentrations are prepared, the working volume of the calibration solution is 2-10 μ l, and then 100-200 μ l of reagent R1 and 50-100 μ l of reagent R2 are added. And (3) detecting the main wavelength of 340nm, the sub wavelength of 405nm and the change rate of the read absorbance by a speed method, and drawing a calibration curve. The establishment of the calibration curve in the application is completed on Hitachi 7180, but other mainstream models (AU680, Yapek C16000 and the like) are detected to be available.

2. Precision experiment

The high, medium and low quality control products and clinical samples were measured by using the calibration curve established above, and the measurements were performed 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 the D306C mutant)

3. Linear experiment

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

γ＝8.8026x+2.9902

R²＝0.9985

TABLE 3 vancomycin kit (for D306C mutant) linearity data

Sample(s)	Measured value 1	Measured value 2	Measured value 3	Mean value	Theoretical value	Relative deviation of	Absolute deviation
								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％

Detection example 2 anti-interference action against common drugs

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

acetaminophen, phenacetin, cefazolin, furosemide, fluphenazine, kanamycin B, oxytetracycline, hydrochlorothiazide, sisomicin, amikacin, prednisolone, cephalexin, fusidic acid, caffeine, lincomycin, penicillin, ibuprofen, spectinomycin, amphotericin B, prednisone, carbamazepine, gentamicin, cephamide, phenytoin, amithicillin, kanamycin A, heparin, salicylic acid, lorazepine.

Test example 3 airborne stability

The quality control materials with high, medium and low concentrations are selected and detected three times every day or every other day, and data show that the calibration period of the vancomycin detection kit (aiming at the 375 mutant) can be stable for more than two weeks. After the reagent was unsealed, it was placed on the machine and the measurements showed an on-machine stability of over 40 days (table 6).

Detection example 4 Interval Difference between lots of vancomycin detection kit

Three batches of the reagent of the present application (D306C mutant) and the reagent prepared by the control coupling method were used, and the difference in absorbance change between the different batches was calculated by separate calibration.

TABLE 4 inter-batch Difference

Detection example 5 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-vancomycin conjugate, the activity of 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 experimental group with the antibody and an experimental group without the antibody is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.

2. Reaction system

TABLE 5 preparation of reagents for detection of antibody inhibition

3. Results

And (3) comparing the absorbance values of the G6 PDH-vancomycin conjugate when the antibody is added with the antibody and when the antibody is not added, respectively detecting the absorbance values of the G6 PDH-vancomycin conjugate to obtain the inhibition condition of the antibody on G6 PDH.

The antibody inhibition rate was × 100% based on the change in absorbance of G6 PDH-vancomycin with antibody/the change in absorbance of G6 PDH-vancomycin without antibody).

Compared with the published mutation site (A45C), the mutant of the application has obviously improved antibody inhibition rate which can reach more than 35 percent (G426C: 35%; D375C: 50%) and can reach 56 percent (D306C). Whereas the inhibition rates of previously published mutation sites (e.g. a45C, K55C) were 32% and 41%.

While not being bound to a particular theory, it may be partially explained as: compared with the G6PDH mutant (A45C, K55C) in the prior art, the mutation site (i.e. the site for introducing free sulfydryl) in the enzyme mutant of the application is the site for coupling with hapten (such as hormone, small molecule drug and the like). When the hapten binds to a hapten-specific antibody at this position, the steric hindrance formed has minimal effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it does not substantially affect the steric folding of the molecule. Therefore, the position of this mutation site is very important, and needs to be compatible with the activity of G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The enzyme mutant has obviously improved antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the vancomycin is prepared into the kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, specificity and the like.

Sequence listing

<110> Beijing Jiuqiang Biotechnology Ltd

<120> 6-phosphoglucose 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

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<211>486

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<213> Leuconostoc pseudomesenteroides (L eutonostoc pseudomesenteroides)

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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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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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 was 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, D at position 375 was replaced 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, G at position 426 was replaced 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

GlyTrp 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 of a glucose-6-phosphate dehydrogenase mutant and a hapten in a molar ratio of 1: 1 is coupled;

the hapten is vancomycin or a derivative thereof;

preferably, the vancomycin derivative is represented by formula I:

wherein,

m is an integer of 1 to 10, preferably 1 to 5.

2. The conjugate of claim 1, wherein:

the glucose-6-phosphate dehydrogenase mutant comprises a mutation selected from the group consisting of: D306C, D375C, G426C;

preferably, the glucose-6-phosphate dehydrogenase mutant is represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4.

3. A reagent comprising the conjugate of claim 1 or 2.

4. Use of a conjugate according to claim 1 or 2 in the preparation of a detection reagent, wherein:

the detection reagent is a vancomycin detection reagent;

preferably, the detection reagent is selected from: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

5. Use of a conjugate according to claim 1 or 2 for the preparation of a detection device:

the detection device is a detection device for vancomycin;

the detection device is selected from any one of the following forms: pore plate, particle, chip and test paper;

preferably, the test device is used for homogeneous immunoassay of vancomycin.

6. A vancomycin detection kit, comprising:

a first reagent comprising a substrate, a vancomycin antibody, a buffer;

a second reagent comprising the conjugate of claim 1 or 2, a buffer;

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

optionally, a quality control comprising 10mM to 500mM buffer, 2 μ g/ml to 60 μ g/ml vancomycin.

7. The vancomycin detection kit of claim 6, comprising:

a first reagent comprising:

10mM to 500mM, preferably 50mM to 300mM, buffer,

5mM to 50mM, preferably 10mM to 20mM, glucose-6-phosphate,

5mM to 50mM, preferably 10mM to 20mM, oxidized β -nicotinamide adenine dinucleotide,

0.5 mg/L to 5.0 mg/L, preferably 1.0 mg/L to 3.0 mg/L anti-phenobarbital antibodies,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L, stabilizer,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L of surfactant,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L preservative;

a second reagent comprising:

10mM to 500mM, preferably 50mM to 300mM, buffer,

The conjugate of claim 1 or 2, from 10ng/m L to 500ng/m L, preferably from 50ng/m L to 200ng/m L,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L, stabilizer,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L of surfactant,

0.1 g/L to 5 g/L, preferably 1 g/L to 5 g/L preservative;

the buffer is selected from one or a combination of the following: TAPS buffer solution, phosphate buffer solution, glycine buffer solution, Tris buffer solution, boric acid buffer solution, MOPS buffer solution and HEPES buffer solution;

preferably, the buffer of the first reagent is a TAPS buffer;

preferably, the buffer of the second reagent is Tris buffer;

the pH of the buffer is 7 to 8;

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; preferably bovine serum albumin;

the surfactant is selected from one or a combination of the following: brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20;

the preservative is selected from one or a combination of the following: azide, MIT, biological preservative PC, thimerosal;

preferably, the preservative is selected from the group consisting of: sodium azide, lithium azide and PC-300.

8. The vancomycin detection kit of claim 6, comprising:

a first reagent comprising:

50mM TAPS buffer, pH 8.0,

15mM glucose-6-phosphate,

15mM oxidized β -nicotinamide adenine dinucleotide,

2.0 mg/L vancomycin antibody,

1 g/L bovine serum albumin,

1g/L Tween20、

1 g/L sodium azide;

a second reagent comprising:

50mM Tris buffer, pH 8.0,

100ng/m L of the conjugate of claim 1 or 2,

1 g/L bovine serum albumin,

1g/L Tween20、

1 g/L sodium azide.

9. A method of preparing a conjugate comprising the steps of:

1) providing vancomycin or a derivative thereof;

2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2;

3) the glucose-6-phosphate dehydrogenase mutant is coupled with the vancomycin or the derivative thereof;

the vancomycin derivative is shown in a formula I:

wherein,

m is an integer of 1 to 10, preferably 1 to 5;

preferably, the method for preparing the conjugate comprises the following steps:

1) providing a vancomycin derivative, preferably in an aprotic solvent;

2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2, preferably in a buffer;

3) contacting the glucose dehydrogenase-6-phosphate mutant and the vancomycin derivative at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that the vancomycin derivative and the glucose dehydrogenase-6-phosphate mutant are coupled to obtain the conjugate;

4) optionally, purifying, preferably desalting, the conjugate;

step 1) and step 2) are interchangeable or parallel;

the buffer is selected from: PBS, Tris, TAPS, TAPSO,

the buffer pH is 6.0 to 8.0;

the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide;

preferably, prior to step 3), the glucose-6-phosphate dehydrogenase mutant comprises a free thiol group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375 or 426.