CN116577495A

CN116577495A - Method for preparing conjugate

Info

Publication number: CN116577495A
Application number: CN202310726230.5A
Authority: CN
Inventors: 龚俊; 王贵利; 张启飞; 李垚艳; 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: 2023-08-11
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 present application relates to a process for the preparation of conjugates. 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

Method for preparing conjugate

The application relates to a Chinese patent application ' 6-phosphoglucose dehydrogenase mutant ' filed on 1 month and 8 days in 2020 and a divisional application of ' 6-phosphoglucose dehydrogenase mutant ' and application of mutant in preparation of amikacin detection reagent ' (application number 2020100173769).

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 an amikacin 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.

Amikacin (Amikacin) has the structural formula shown below:

amikacin is an amino glycoside antibiotic and is used for treating infections of urinary tract, lower respiratory tract, abdominal cavity, soft tissue, bone, joint, reproductive system and other parts, septicemia and the like caused by gram-negative bacilli. It can cause irreversible ototoxic effect, has very little renal toxicity, is reversible, and has rare neuromuscular blockade.

Therefore, attention is paid to monitoring adverse drug reactions. And because of differences of individual metabolisms of the drugs, the drug administration method should be combined with blood concentration monitoring during clinical use to formulate a reasonable dosing scheme so as to avoid adverse reactions as much as possible.

Currently known amikacin detection methods mainly comprise: high Performance Liquid Chromatography (HPLC), chemiluminescence immunity, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, latex agglutination turbidimetry, 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 methods rely on activation of reactive groups carried by the small molecule drug (amikacin) itself prior to reaction with the enzyme. Such coupling methods can occur when multiple amikacin 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 preparation of amikacin 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., amikacin) 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: theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including digoxin, digitoxin), zymophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the product of the uronictin metabolite, cotinine, uromorphine, uromonohydric phenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropin, 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 amikacin or a derivative thereof.

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

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

wherein, the liquid crystal display device comprises a liquid crystal display device,

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.

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

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 an amikacin detection reagent.

According to some embodiments, there is provided the use of a conjugate of the application in the preparation of an amikacin 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 an amikacin 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 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 application and a buffer;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0 μg/ml to 50 μg/ml amikacin (e.g. 0, 3, 5, 10, 20, 30, 35, 40, 45, 50 μg/ml or any value in between); and

-optionally, a quality control comprising 10mM to 500mM buffer, 3 μg/ml to 40 μg/ml (e.g. 3, 4, 5, 10, 20, 30, 40 μg/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,

5mM to 50mM substrate,

0.01 to 10. Mu.g/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. Mu.g/ml),

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,

0.01. Mu.g/ml to 10. Mu.g/ml of the 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. Mu.g/ml),

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 amikacin antibody is derived from: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans.

In some specific embodiments, the amikacin 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 an amikacin 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 glucose 6-phosphate dehydrogenase mutant and the amikacin derivative are mixed according to amikacin derivatives: enzyme = 500:1 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 in between, preferably 2 hours to 3 hours) such that the amikacin derivative and the glucose 6-phosphate dehydrogenase mutant are coupled to give the seed conjugate;

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

In some embodiments, the hapten and enzyme are contacted in 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 thereof; preferably 50.

In some embodiments, the hapten and enzyme contact molar ratio n 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 thereof; preferably 50. 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 amikacin.

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. 1G 6PDH (wild type) amino acid sequence (SEQ ID No. 1); is derived from Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides.

FIG. 2G 6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3G 6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 4G 6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

EXAMPLE 1 Synthesis of amikacin derivative

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

Through mass spectrum and nuclear magnetism identification, the amikacin derivative has correct structure. This example allows amikacin to have a group that can bind to enzymes.

EXAMPLE 2 coupling of amikacin derivative to G6PDH molecule

1. The coupling method of the application

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

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

g6pdh solution: g6PDH mutant was dissolved in PB 100mmol, naCl 100mmol,pH 8.0,6mg/ml;

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 amikacin 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-amikacin conjugate.

2. Control coupling method

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

dropwise adding 10-200 mM sodium periodate 5-15 mL into the solution, slightly oscillating, and stirring at room temperature for reaction for 0.5-2 hours;

dripping 0.5 to 2M glycol 0.5 to 1mL, and stirring at room temperature for reaction for 5 to 10 minutes;

dropwise adding the reaction mixture into 5-15 mL of 2-3% G6PDH solution under stirring, regulating the pH of the solution to 9.0-9.5, continuing stirring for reaction for 0.5-2 hours, and stabilizing the pH of the solution;

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

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

EXAMPLE 3 preparation of the kit

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

reagent R1 comprising:

tris buffer 100mM, pH 7.0

10mM glucose 6-phosphate

10mM beta-nicotinamide adenine dinucleotide

Amikacin antibody (commercially available antibody, not particularly limited) at 0.5. Mu.g/ml

1g/L bovine serum albumin

1g/L Tween80

1g/L sodium azide;

reagent R2, comprising:

MES buffer 200mM, pH8.0

0.1 μg/ml G6 PDH-amikacin conjugate

100mM NaCl

1g/L bovine serum albumin

1g/L Tween80

1g/L sodium azide;

calibration material: 20mM HEPES buffer, 0 μg/ml, 3 μg/ml, 10 μg/ml, 20 μg/ml, 35 μg/ml, 50 μg/ml amikacin (or added as needed);

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

The reagent (optionally containing quality control materials and calibrator) is assembled into the amikacin homogeneous enzyme immunoassay kit.

Test case

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6 PDH-amikacin) competes with non-labeled antigen (amikacin) for binding with quantitative antibody (amikacin antibody), and when the more the antibody is bound with non-labeled antigen, the more activity the enzyme-labeled antigen releases, and the more NAD+ which is a substrate is catalyzed by the enzyme to generate NADH.

Detecting the absorbance change of NADH at 340nm wavelength to calculate the amikacin content in the liquid.

TABLE 1 full automatic Biochemical instrument parameters

Detecting machine type	Yaban C16000
		Analysis/time/read point	Rate/10 min/28-33
R1/R2/S	150：50：3
		Wavelength (auxiliary/main)	405/340
Reaction type	Incremental increases
		Calibration type	Spine
Calibration point	6
		Concentration of calibrator	0/3/10/20/35/50

Test example 1. Performance of the kit of the application

1. Calibration experiment

TABLE 2 Amikacin detection kit calibration absorbance

2. Precision experiments

TABLE 3 Total imprecision

3. Repeatability of

TABLE 4 repeatability

4. Recovery test

TABLE 5 recovery data

5. Linear experiments

TABLE 6 linearity

/>

Test example 2 acceleration stability

The reagent (G426 mutant) has the advantages that the calibrated absorbance is reduced by less than 5% after the reagent is accelerated for 7 days at 37 ℃, and the calibrated absorbance is obviously reduced after the reagent is accelerated for 7 days at 37 ℃ in comparison with the reagent.

TABLE 7 accelerated stability at 37℃

Detection example 3 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-amikacin 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, wherein the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. Reaction system

TABLE 8 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-amikacin 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 = (1-absorbance change for G6 PDH-amikacin conjugate with antibody/absorbance change for G6 PDH-amikacin without antibody) ×100%.

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

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 amikacin is prepared into a kit, the reagent has obvious performance improvement in the aspects of the inter-batch variation coefficient, linearity, repeatability, stability and the like.

Claims

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

1) Providing amikacin derivatives;

2) Providing a glucose 6-phosphate dehydrogenase mutant;

3) The glucose-6-phosphate dehydrogenase mutant is coupled with the amikacin derivative;

the amikacin derivative is shown in a formula I:

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

the glucose 6-phosphate dehydrogenase mutant comprises a D306C or D375C mutation compared to the wild-type glucose 6-phosphate dehydrogenase: the method comprises the steps of carrying out a first treatment on the surface of the

The glucose 6-phosphate dehydrogenase mutant is shown as SEQ ID No.2 or SEQ ID No. 3.

2. The method according to claim 1, comprising the steps of:

1) Providing an amikacin derivative, preferably in an aprotic solvent;

2) Providing a glucose 6-phosphate dehydrogenase mutant, preferably providing said glucose 6-phosphate dehydrogenase mutant in a buffer;

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

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

step 1) and step 2) may be interchanged or parallel;

the buffer is selected from one or a combination of the following: PBS, tris, TAPS, TAPSO the number of the individual pieces of the plastic,

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 has a free thiol group at position 306 or 375.