CN111504921B - 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of gentamicin detection reagent - Google Patents

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a gentamicin 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, G C. The gentamicin detection kit prepared by using the glucose-6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenience in operation, short detection time, small batch difference and good application prospect.

Description

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

Priority of application No. 201910017764.4 filed on 9/1/2019 and application No. 201910423122.4 "glucose-6-phosphate dehydrogenase mutant and use thereof in the preparation of a test agent" filed on 21/5/2019, which are hereby incorporated 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 gentamicin detection kit.

Background

Haptens, certain small molecular substances (molecular weight less than 4000 Da), alone are not capable of inducing an immune response, i.e. are not immunogenic, but can gain immunogenicity when cross-linked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysines, inducing 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 conjugated 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 with specific structures are called simple haptens.

Small molecule antigens or haptens are not able to be measured by the double antibody sandwich method because they lack more than two sites that can be used in the sandwich method, and the competition mode is mostly adopted. 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 amount of antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color development is. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.

Gentamicin (Gentamicin) is a mixture of three compounds, the structures of which are shown below:

gentamicin, which was first discovered by Weinstein in 1963, is a multicomponent aminoglycoside antibiotic produced by micromonospora, and includes C1, C2, C1a, C2a, and C2b components. Gentamicin is widely used clinically as its main components C1, C2 and C1a, of which the highest antibacterial activity is C1a, which is a precursor for the synthesis of etimicin, and secondly C2b, which is also known as saxamycin. Such antibiotics can bind to 16S rRNA on the 30S subunit of the bacterial ribosome, causing misreading of the genetic code and thereby blocking the synthesis of bacterial proteins, and are therefore used primarily for the treatment of bacterial infections, especially infections caused by gram-negative bacteria.

Gentamicin is an aminoglycoside that acts on ribosomes in bacteria, inhibits bacterial protein synthesis, and destroys the integrity of bacterial cell membranes. The absorption is rapid and complete after intramuscular injection of gentamicin. After local washing or local external application, a certain dosage can be absorbed by the body surface. Administration via the eye rarely absorbs into intraocular tissues or into the systemic blood circulation. It is absorbed little after oral administration. After intramuscular injection or intravenous drip, the blood concentration reaches the peak value in 30-60 minutes, and the average blood concentration peak value is about 4 mu g/ml. The protein binding rate of gentamicin is very low, and the drug is mainly distributed in extracellular fluid after being absorbed. The half-life of the adult is 2-3 hours, and the half-life of patients with fever, anemia and serious burn or patients with combined carbenicillin can be shortened. Gentamicin is not metabolized in the body and is mainly excreted via glomerular filtration with urine.

Currently, the known methods for detecting gentamicin mainly comprise: enzyme-linked immunosorbent assay, chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, these detection methods have many defects, for example, although chemiluminescence has good sensitivity, a special device is required, and the cost for use is high, which is not favorable for popularization. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex enhanced immunoturbidimetry are mainly used for detection.

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

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

The prior art (for example, but not limited to CN 108107203A) describes a gentamicin derivative-G6 PDH conjugate and its preparation method:

1) Weighing G6PDH, and dissolving in a PBS buffer solution at room temperature;

2) Dissolving a certain amount of gentamicin, 1-ethyl-3-carbodiimide and N-hydroxy thiosuccinimide in a Mes solution, and stirring and dissolving for 15-60min at room temperature for activation;

3) Dropwise adding the activated gentamicin solution into the dissolved G6PDH, and stirring for dissolving;

4) Stirring and dissolving at 2-8 ℃ overnight;

5) Purifying the coupled enzyme-labeled antigen to obtain a glucose dehydrogenase-gentamicin conjugate, and storing at 2-8 ℃.

However, the prior art methods rely on the activation of reactive groups carried by the small molecule drug itself, followed by reaction with an enzyme. Such a strategy makes it difficult to ensure orientation between small molecule drugs and enzymes 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 preparing a gentamicin detection kit.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the already published mutant of glucose-6-phosphate dehydrogenase of patent US006090567a (homogenomic 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, D375C, G C.

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 and 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: x is coupled. In some embodiments, x is 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 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., gentamicin) that do not themselves carry a coupling group (e.g., a group that reacts with a sulfhydryl group) can be engineered to carry a linker for covalent binding to a sulfhydryl 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: theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxigenin), mycophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites cotinine of uronicotin, uromorphine, derivatives of uromonophenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormones, prolactin, placental prolactin, growth hormones, follicle stimulating hormones, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, retro-triiodothyronine, free thyroxine, free triiodothyronine, cortisol urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfates, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 α hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandin, plasma 6-one prostaglandin F1 α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, 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 gentamicin or a derivative thereof.

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

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

in some embodiments, m is an integer from 0 to 20, preferably from 1 to 10, preferably from 1 to 6, e.g., 1, 2, 3, 4, 5, 6.

In some embodiments, X is maleimide, bromoacetyl, vinyl sulfone, or aziridine.

The skilled person will appreciate that X functions to react with the thiol group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine, and thiol groups is contemplated. Although specific groups are employed in the examples, they are not intended to be limiting.

In some specific embodiments, the gentamicin derivative has a structure selected from the group consisting of:

m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6.

In some specific embodiments, the gentamicin derivative has a structure selected from the group consisting of:

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 gentamicin detection reagent.

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

-a first reagent comprising a substrate, a buffer and a gentamicin 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 10 μ g/ml gentamicin; and

-optionally, a quality control comprising 10mM to 500mM buffer, 0.5 μ g/ml to 8 μ g/ml gentamicin.

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

0.1 to 10 mug/ml of gentamicin antibody,

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,

0.1. Mu.g/ml to 10. Mu.g/ml of a conjugate according to the application,

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: 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 solution is 10mmol/L to 500mmol/L, preferably 100mM; 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 and polyethylene glycol 8000; bovine serum albumin is preferred.

In some embodiments, the surfactant is selected from one or a combination of: brij23, brij35, triton X-100, triton X-405, tween20, tween30, tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

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 gentamicin antibody is derived from: mouse, rat, cat, dog, primate, bovine, equine, ovine, camelid, avian, human.

In some specific embodiments, the gentamicin 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 a gentamicin 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) The glucose-6-phosphate dehydrogenase mutant and the gentamicin derivative (e.g., in a molar ratio of 1: n) contacting for 1 hour to 4 hours (preferably 2 hours to 3 hours) to allow the gentamicin derivative and the glucose-6-phosphate dehydrogenase mutant to couple to obtain the seed conjugate;

4) The conjugate is optionally purified, for example, desalted 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 120, 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, 60, 70, 80, 90, 100, 110, 120; preferably 20 to 40.

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

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

Wild-type glucose-6-phosphate dehydrogenase does not contain a free thiol group, and thus in some embodiments, the glucose-6-phosphate dehydrogenase is genetically engineered to have a free thiol group with a mutation of 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 is the amino acid sequence of G6PDH (D306C) (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 Gentamicin derivatives

Wherein m is 1.

Gentamicin (100mg, 0.21mmol) and Compound 1 (64mg, 0.21mmol) were dissolved in 5mL of water and stirred at room temperature (18-28 deg.C, preferably 20-25 deg.C) for 5h. Direct HPLC separation gave the gentamicin derivative (110mg, 78%). The structure of the product was confirmed by a conventional method. This example allows gentamicin to carry a group that can bind to the enzyme.

Example 2 coupling of Gentamicin derivatives to G6PDH molecules

1. Test method of the present application

The G6 PDH-gentamicin conjugates according to the present application were coupled as follows: a thiol-reactive group (such as, but not limited to, a maleimide group) on a gentamicin derivative molecule is covalently bound to a thiol on a G6PDH molecule.

1. The gentamicin derivative prepared in example 1 was dissolved in DMF (10 mg/ml);

2. providing a glucose 6 phosphate dehydrogenase solution (5 mg/mL enzyme, 100mmol PB, 100mmol nacl, ph = 8.0);

3. shaking 2ml glucose 6 phosphate dehydrogenase solution, 7.5ml PB solution and 0.5ml gentamicin derivative solution at room temperature (18-28 deg.C, preferably 20-25 deg.C) for 4 hr;

4. desalting column treatment (100 mM PB of desalting solution, 0.1% NaN% ₃ 1% nacl, ph = 8.0), the protein peak was collected to obtain G6 PDH-gentamicin conjugate.

2. Control coupling method

The glucose-6-phosphate dehydrogenase-gentamicin conjugate was prepared according to the method of CN108107203A and stored at 2 to 8 ℃.

Example 3 preparation of the kit

A kit for detecting gentamicin was prepared, which contained:

a reagent R1 comprising:

50mM HEPES，pH 7.0

10mM glucose-6-phosphate

10mM beta-nicotinamide adenine dinucleotide

Gentamicin antibody 1. Mu.g/ml (commercially available antibody, without particular limitation)

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

a reagent R2 comprising:

200mM Tris buffer, pH 8.0

G6 PDH-gentamicin conjugate of 1 ug/ml

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration products: 20mM HEPES buffer, and 0.0, 0.5, 1.5, 3.0, 6.0, 10.0. Mu.g/ml gentamicin (or added as needed);

quality control product: 20mM HEPES buffer, and 2.0. Mu.g/ml, 4.0. Mu.g/ml, 7.5. Mu.g/ml gentamicin (or added as needed).

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

Example of detection

TABLE 1 parameters of fully automatic biochemical analyzer

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

TABLE 2 accuracy, precision (for D306C mutant)

TABLE 3 Linearity (for D306C mutant)

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Detection example 2 anti-interference of common drugs

The following compounds were selected as interferents and the gentamicin calibrator was determined in the presence of the interferents at the concentrations shown in table 4, showing no statistically significant interference.

TABLE 4 determination of interference resistance (for D306C mutant)

Detection example 3 correlation

1. Test method

80 samples of fresh serum were taken, each divided into two aliquots, each at a volume of not less than 500. Mu.l, and one of the samples was assayed twice on Hitachi 7180 apparatus using the reagents of the present application (for the D306C mutant) and the other sample was assayed using Shimadzu HPLC. The two method values were analyzed for correlation using scatter plots.

2. And (3) test results:

the resulting function is y =1.0009x-0.0132, correlation coefficient R ² ＝0.9963。

The result shows that the reagent has good correlation with the concentration value of gentamicin in the sample measured by HPLC (visible as gold standard).

TABLE 5 correlation analysis (unit: ng/ml)

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Detection example 4 batch to batch variation of gentamicin 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 differences in absorbance change between the different batches were calculated by separate calibration.

TABLE 6 calibration data between batches

TABLE 7 comparison of lots

Detection example 5 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-gentamicin 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 assay reagent for antibody inhibition

3. Results

And (3) comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance values of the G6 PDH-gentamicin conjugate to obtain the inhibition condition of the antibody on G6 PDH.

Antibody inhibition = (1-absorbance change of G6 PDH-gentamicin with antibody/absorbance change of G6 PDH-gentamicin without antibody) × 100%.

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: 48%) and can reach as high as 50 percent (D306C). Whereas the inhibition of the previously published mutation sites (e.g. A45C, K C) was 32% and 37%.

While not being bound to a particular theory, it may be explained in part as: compared with the G6PDH mutant (A45C, K C) in the prior art, the mutant site (i.e., the site for introducing free thiol) in the enzyme mutant of the present application is the site for coupling with hapten (such as hormone, small molecule drug, etc.). 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 gentamicin is prepared into the kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, specificity and the like.

Test example 6 alternative

Referring to the preparation method of example 3, different test kits and control kits were respectively configured, except that the kit prepared in example 3 was replaced as follows:

scheme 1: the buffer in the first reagent and the second reagent is replaced by phosphate buffer, glycine buffer, boric acid buffer, or MOPS buffer in the range of 50 to 100mM pH 7.0-8.0;

scheme 2: the stabilizer in the first reagent and the second reagent is replaced by 0.5 to 2.5g/L of trehalose, sucrose, mannitol or polyethylene glycol 6000;

scheme 3: the surfactant in the first and second reagents is replaced with 0.5 to 2.5g/L Triton X-100, tween80, brij35, or Brij 23;

scheme 4: the preservative in the first reagent and the second reagent is replaced by lithium azide or PC-300;

scheme 5: the compound of formula II is replaced by the compounds of formulae III, IV and V.

The test kit and the control kit of three different batches in each scheme are tested according to the method of the detection example 4, the comparison result is close to that in tables 6 to 7, and the difference variation between the batches of the test kit is smaller than that of the control kit (data not shown).

Sequence listing

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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 replaced by C compared with wild type

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims (4)

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

the gentamicin derivative has a structure shown in a formula I:

the compound of the formula I is shown in the specification,

wherein

m is an integer of 0 to 20;

x is selected from: maleimide, bromoacetyl, vinyl sulfone, or aziridine;

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

the glucose-6-phosphate dehydrogenase mutant is any one of the following sequences: SEQ ID No.2 and SEQ ID No.3.

2. The conjugate of claim 1, wherein:

m is an integer of 1 to 10.

3. The conjugate of claim 1, wherein:

m is an integer of 1 to 6.

4. A reagent comprising the conjugate of any one of claims 1 to 3.

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