CN111537452A

CN111537452A - 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of amikacin detection reagent

Info

Publication number: CN111537452A
Application number: CN202010017376.9A
Authority: CN
Inventors: 王贵利; 张启飞; 李垚艳; 龚俊; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2020-01-08
Publication date: 2020-08-14
Anticipated expiration: 2040-01-08
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 6-phosphoglucose dehydrogenase mutant and application thereof in preparing an amikacin 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-glucose phosphate dehydrogenase mutant and application thereof in preparation of amikacin 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 an amikacin 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.

Small molecule antigens or haptens lack two or more sites that can be used in sandwich assays, and therefore cannot be measured using the double antibody sandwich assay, and often use a competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete to bind with the solid-phase antibody. The more the amount of the antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color develops. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.

Amikacin (Amikacin) has the following structural formula:

amikacin is aminoglycoside antibiotic, and can be used for treating infections of urinary tract, lower respiratory tract, abdominal cavity, soft tissue, bone, joint, reproductive system, etc. caused by gram-negative bacillus, and septicemia. This herb can cause irreversible ototoxic effect, and has very slight renal toxicity, reversible property, and rarely blocked nerve and muscle.

Therefore, attention is paid to the monitoring of adverse reactions of the medicine. Moreover, due to differences of drug metabolism individuals, a reasonable administration scheme is formulated by combining blood concentration monitoring during clinical use, so that adverse reactions are avoided as much as possible.

The currently known amikacin detection methods mainly comprise: high Performance Liquid Chromatography (HPLC), chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, latex agglutination turbidimetry, etc. The HPLC method needs complex sample pretreatment, has complex operation and long period and is expensive in cost; the reagent of the luminescence immunoassay method has high cost, is not suitable for the detection of conventional treatment drugs, 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 methods rely on the activation of reactive groups carried by the small molecule drug (amikacin) itself, followed by reaction with an enzyme. Such a conjugation method may lead to the situation that a plurality of amikacin are linked to the same glucose hexaphosphate dehydrogenase, and it is difficult to ensure the consistency of the conjugation site, and it is difficult to ensure the 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 need in the art, the present application provides a novel mutant of glucose-6-phosphate dehydrogenase and its use in the preparation of amikacin 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., amikacin) that do not themselves bear a coupling group (e.g., a group that reacts with a thiol group) can be engineered to have 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 exemplified by, but not limited to: theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin, digoxigenin), mycophenolic acid, rapamine, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, cotinine, uromorphine, derivatives of urotensin, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, erythropoietin, adrenocorticotropic hormone, luteinizing hormone, diuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosterol, and/or, Urinary 17-ketosteroids, dehydroepiandrosterone and sulfate esters, aldosterone, urovanillyl 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-one prostaglandin F1 alpha, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, sodium, adenosine cyclophosphate, guanosine cyclophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

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

In particular embodiments, the hapten is an amikacin derivative bearing a thiol-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

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

wherein the content of the first and second substances,

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

In some specific embodiments, the amikacin derivative has the structure shown in formula II:

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 the preparation of an amikacin detection reagent.

According to some embodiments, there is provided a use of a conjugate of the present application in the preparation of an amikacin 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 an amikacin 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 an amikacin detection kit comprising:

-a first reagent comprising a substrate, a buffer and an amikacin 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 50 μ g/ml amikacin (e.g., 0, 3, 5, 10, 20, 30, 35, 40, 45, 50 μ g/ml or any value in between); and

-optionally, a quality control comprising 10mM to 500mM buffer, 3 μ g/ml to 40 μ g/ml (e.g. 3, 4, 5, 10, 20, 30, 40 μ g/ml or any value in between) amikacin.

According to one embodiment, there is provided an amikacin detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

0.01 to 10 mug/ml amikacin antibody (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, 2, 3, 4, 5 mug/ml),

0.1 to 5g/L stabilizer,

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

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

0.01 to 10 mug/ml of a conjugate according to the application (0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mug/ml),

0.1 to 5g/L 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: TAPS, tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 50 to 100 mM; the pH of the buffer is 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: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.

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

In some specific embodiments, the amikacin antibody is selected from the group consisting of: 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 an amikacin 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) reacting the glucose-6-phosphate dehydrogenase mutant and the amikacin derivative at 18 ℃ to 28 ℃ in accordance with the ratio of amikacin derivative: 500 parts of enzyme: 1 to 1: contacting at a molar ratio of 500 (preferably 50: 1 to 1: 50) for 1 hour to 4 hours (1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or any value therebetween, preferably 2 hours to 3 hours) to allow conjugation of the amikacin derivative and the glucose-6-phosphate dehydrogenase mutant to occur, thereby obtaining the conjugate;

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

In some embodiments, the hapten and the enzyme are contacted in the reaction system at a molar ratio of 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; preferably 50.

In some embodiments, the contact molar ratio n of hapten to enzyme in the reaction system: 1, 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; preferably 50. 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 amikacin.

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.G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides of Leuconostoc.

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

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

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

Detailed Description

Examples

Example 1 Synthesis of Amikacin derivatives

Amikacin (123mg, 0.21mmol) and compound 1(64mg, 0.21mmol) were dissolved in 5mL of water and stirred at room temperature (18-28 ℃ C.) for 5h, according to the above scheme. HPLC separation gave the amikacin derivative (100mg, 61%).

The amikacin derivative has correct structure through mass spectrum and nuclear magnetic identification. This example allows amikacin to carry a group that can bind to the enzyme.

Example 2 coupling of Amikacin derivatives to G6PDH molecules

First, the coupling method of the present application

The coupling of the G6 PDH-amikacin conjugate according to the application was performed as follows: a thiol-reactive group (such as, but not limited to, a maleimide group) on an amikacin derivative molecule is covalently bound to a thiol on a G6PDH molecule.

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

g6PDH solution: dissolving the G6PDH mutant in PB 100mmol, NaCl 100mmol, pH8.0 and 6 mg/ml;

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 N, N-dimethylformamide solution of amikacin derivative was added thereto;

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

Two, control coupling method

Accurately weighing 100-300 mg amikacin, and dissolving with 5-15 mL of absolute ethyl alcohol;

5 to 15mL of 10 to 200mM sodium periodate is added into the solution dropwise, and the solution is slightly shaken and stirred for reaction for 0.5 to 2 hours at room temperature;

0.5 to 2M of glycol is dripped into 0.5 to 1mL of the mixture, and the mixture is stirred and reacts for 5 to 10 minutes at room temperature;

dropwise adding the reaction mixture into 5 to 15mL of a2 to 3 percent G6PDH solution which is stirring, adjusting the pH of the solution to 9.0 to 9.5, continuing stirring for reaction for 0.5 to 2 hours, and stabilizing the pH of the solution;

adding 100-200 mg of sodium tetrahydroborate, and stirring and reducing for 12-24 hours;

purifying by a G-25 gel chromatography column to obtain the G6 PDH-amikacin conjugate.

Example 3 preparation of the kit

Preparing the following kit for detecting amikacin comprising:

reagent R1, comprising:

tris buffer 100mM, pH 7.0

10mM glucose-6-phosphate

10mM beta-nicotinamide adenine dinucleotide

0.5. mu.g/ml amikacin antibody (commercially available antibody, without particular limitation)

1g/L bovine serum albumin

1g/L Tween80

1g/L sodium azide;

reagent R2, comprising:

MES buffer 200mM, pH8.0

G6 PDH-amikacin conjugate at 0.1. mu.g/ml

100mM NaCl

1g/L bovine serum albumin

1g/L Tween80

1g/L sodium azide;

calibration products: 20mM HEPES buffer, and 0. mu.g/ml, 3. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, 35. mu.g/ml, 50. mu.g/ml amikacin (or added as needed);

quality control product: 20mM HEPES buffer, and 4-5. mu.g/ml, 14-16. mu.g/ml, 28-32. mu.g/ml amikacin (or added as needed).

And assembling the reagents (optionally containing quality control products and calibration products) into the amikacin 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-amikacin) and a non-labeled antigen (amikacin) compete for binding with a quantitative antibody (amikacin antibody), and the more the antibody binds to the non-labeled antigen, the more activity is released from the enzyme-labeled antigen, and the more NADH is generated from an enzyme-catalyzed substrate NAD +.

And detecting the absorbance change of NADH under the wavelength of 340nm to calculate the content of amikacin in the liquid.

TABLE 1 parameters of fully automatic biochemical analyzer

Detecting machine type	Yapei C16000
		Analysis/time/read Point	Speed/10 min/28-33
R1/R2/S	150：50：3
		Wavelength (auxiliary/main)	405/340
Type of reaction	Incremental increase
		Calibration type	Spine
Calibration point	6
		Concentration of calibrator	0/3/10/20/35/50

Test example 1 Performance of the kit of the present application

1. Calibration experiment

TABLE 2 Amikacin assay kit calibration Absorbance

2. Precision experiment

TABLE 3 Total inaccuracy

3. Repeatability of

TABLE 4 repeatability

4. Recovery testing

TABLE 5 recovery data

5. Linear experiment

TABLE 6 linearity

Test example 2 acceleration stability

After the reagent (G426 mutant) is accelerated at 37 ℃ for 7 days, the reduction of the calibration absorbance is less than 5%, and after the contrast reagent is accelerated at 37 ℃ for 7 days, the reduction of the calibration absorbance is obvious.

TABLE 7.37 ℃ accelerated stability

Test example 3 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-amikacin 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 added and an experimental group without the antibody added is compared by detecting the change of NADH amount, and the difference is reflected in the inhibition capacity of the antibody on G6 PDH.

2. Reaction system

TABLE 8 preparation of reagents for measuring antibody inhibition

3. Results

And (3) comparing the absorbance values of the G6 PDH-amikacin 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-amikacin conjugate to obtain the inhibition condition of the antibody on the G6 PDH.

The antibody inhibition rate was (1-absorbance change of G6 PDH-amikacin conjugate with antibody/absorbance change of G6 PDH-amikacin without antibody) × 100%.

Compared with the published mutation site (A45C), the mutant of the application has obvious improvement on the retention of enzyme activity, and can reach more than 39 percent (G426C: 39%; D375C: 48%) and up to 60 percent (D306C). Published mutation sites (e.g. a45C, K55C) were prepared as G6 PDH-amikacin conjugates with only 32% and 38% inhibition, according to the methods of the present application.

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 the greatest effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it cannot 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 amikacin is prepared into a kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, repeatability, stability and the like.

Sequence listing

<110> Beijing Jiuqiang Biotechnology Ltd

<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of amikacin detection reagent

<130>390267CG

<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 pseudomesenteroides (Leuconostoc pseudosensoides)

<400>1

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 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

245250 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

405410 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 135140

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 295300

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

the hapten is amikacin or a derivative thereof;

preferably, the amikacin derivative is represented by formula I:

wherein the content of the first and second substances,

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

more preferably, the amikacin derivative is represented by formula II:

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 amikacin 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 amikacin;

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 amikacin.

6. An amikacin detection kit comprising:

a first reagent comprising a substrate, an amikacin 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 50 μ g/ml amikacin; and

optionally, a quality control comprising 10mM to 500mM buffer, 3 μ g/ml to 40 μ g/ml amikacin.

7. The amikacin 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 form beta-nicotinamide adenine dinucleotide,

0.01 to 10. mu.g/ml, preferably 0.1 to 1. mu.g/ml, of amikacin antibody,

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

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

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

a second reagent comprising:

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

The conjugate of claim 1 or 2 in an amount of 0.01 to 10. mu.g/ml, preferably 0.05 to 0.5. mu.g/ml,

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

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

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

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: sodium azide, lithium azide and PC-300.

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

a first reagent comprising:

50mM TAPS buffer, pH8.0,

10mM glucose-6-phosphate,

10mM oxidized beta-nicotinamide adenine dinucleotide,

0.5 mu g/ml amikacin antibody,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide;

a second reagent comprising:

50mM Tris buffer, pH8.0,

0.1. mu.g/ml of the conjugate according to claim 1 or 2,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide.

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

1) providing amikacin 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 amikacin or the derivative thereof;

the amikacin derivative is represented by formula I:

wherein the content of the first and second substances,

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 an amikacin 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-6-phosphate dehydrogenase mutant and the amikacin derivative at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that the amikacin derivative and the glucose-6-phosphate dehydrogenase 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;

the contacting molar ratio of the glucose-6-phosphate dehydrogenase mutant to the amikacin derivative is 50: 1 to 1: 50.