CN111537451B

CN111537451B - Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of tacrolimus detection reagent

Info

Publication number: CN111537451B
Application number: CN202010009570.2A
Authority: CN
Inventors: 张永侠; 封建新; 李薇; 张启飞; 龚俊; 王贵利; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2020-01-06
Publication date: 2023-07-14
Anticipated expiration: 2040-01-06
Also published as: CN111504920A; CN111693473A; CN116699122A; CN111537452B; CN112285037B; CN111504920B; CN116698772A; CN116699125A; CN116679047A; CN111650135B; CN116298257A; CN116381253A; CN116298330A; CN116718761A; CN111487208B; CN116773795A; CN116430056A; CN116338215A; CN116559425A; CN111239060B

Abstract

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

Description

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

The present application claims priority from application numbers 201910017764.4, filed on 1 month 9 in 2019, and 201910423122.4, mutant glucose 6-phosphate dehydrogenase, filed on 5 month 21 in 2019, and their use in preparing detection reagents, which are incorporated herein by reference.

Technical Field

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

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.

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.

Tacrolimus (tacrolimus) as a specific example of hapten has the following structural formula:

tacrolimus, also known as FK506, is a macrolide antibiotic. Tacrolimus was found in japan in 1984 and was first used clinically as an immunosuppressant in 1989. Tacrolimus, an immunosuppressant, has high lipophilicity, incomplete absorption and instability.

The safe and effective therapeutic range of tacrolimus is narrow, and insufficient dosage or too low blood concentration of tacrolimus may cause graft rejection. Too high tacrolimus concentrations can lead to serious adverse effects including nephrotoxicity, neurotoxicity, post-implantation diabetes, increased susceptibility to infection, cancer, hypertension, and gastrointestinal dysfunction.

For the above reasons, tacrolimus blood concentration monitoring is an effective way to assist clinical treatment, improve therapeutic effects, and reduce risk of toxicity.

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

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

The prior art CN108107200a describes a tacrolimus detection kit, wherein a preparation method of a conjugate of glucose-6-phosphate dehydrogenase and tacrolimus is disclosed:

dissolving 20-100mg of tacrolimus in methanol, adding anhydrous sodium acetate, adding carboxymethyl hydroxylamine after dissolving uniformly, dissolving and mixing uniformly, heating under the protection of nitrogen for reacting overnight, then distilling under reduced pressure to obtain wax, adding dimethylformamide for dissolving, filtering to remove precipitate, and distilling under reduced pressure to remove solvent to obtain a product A;

dissolving 10-50mg of product A with 20-100mL of dimethylformamide, slowly dropping 50-150. Mu.L of carbodiimide (EDC) into the solution under stirring, and mixing for 60-150 minutes with rotation;

dissolving 10-50mg of glucose-6-phosphate dehydrogenase with the specification of 100-300KU in PBS buffer solution, and shaking uniformly;

slowly adding the tacrolimus solution into the 3 solution under stirring, and stirring and reacting for 8-16 hours to obtain the conjugate of the glucose-6-phosphate dehydrogenase and the tacrolimus.

However, the prior art methods rely on activation of the reactive groups carried by the small molecule drug (tacrolimus) itself prior to reaction with the enzyme. Such coupling methods can occur when multiple tacrolimus are linked to the same glucose hexaphosphate dehydrogenase, and it is difficult to ensure consistency of the coupling site, and it is difficult 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 present application provides a novel glucose 6-phosphate dehydrogenase mutant and its use in preparing tacrolimus 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 mutants of the present application comprise 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 present application.

According to some embodiments, a host cell is provided 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 which is a glucose 6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: and (3) coupling x.

In some embodiments, x 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 the glucose 6-phosphate dehydrogenase mutant to hapten of the present application 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.

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

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

Hapten such as, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including 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 tacrolimus or a derivative thereof.

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

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

in some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

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

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

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

-a first reagent comprising a substrate, a buffer and a tacrolimus 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, 0ng/ml to 30ng/ml tacrolimus; and

-optionally, a quality control comprising 10mM to 500mM buffer, 5ng/ml to 25ng/ml tacrolimus.

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

a first reagent comprising:

10mM to 500mM buffer,

5mM to 50mM substrate,

0.1 to 10 μg/ml mg/L tacrolimus antibody,

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

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

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer,

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

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

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

0.1g/L to 5g/L preservative;

third reagent: it comprises: the volume ratio of methanol to ethanol is 3:1, and 0.5-5% zinc sulfate.

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: brij23, 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 tacrolimus antibody is derived from: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans.

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

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

1) Providing a tacrolimus 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, said buffer having a pH of 6.0 to 8.0);

3) Contacting the tacrolimus derivative and the glucose 6-phosphate dehydrogenase mutant at a molar ratio n:1 for 1 to 4 hours (preferably 2 to 3 hours) at 18 ℃ to 28 ℃ such that the tacrolimus derivative and the glucose 6-phosphate dehydrogenase mutant are coupled to obtain the seed conjugate;

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

In some embodiments, the contacting molar ratio of enzyme to hapten in the reaction system is 1: n, 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 n is 20 to 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 tacrolimus.

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 pseudoenteroides 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 Tacrolimus derivatives

Where m=1.

Tacrolimus (100 mg,0.11 mmol) was added to a round bottom flask, dissolved in dry DCM (5 mL), to which catalytic equivalent of 4-N, N-lutidine was added, DCC (27 mg,0.13 mmol) was added and stirred under nitrogen until all dissolved. 4-Maleimidobutyric acid (20 mg,0.11 mmol) was added to the reaction system, and stirred at room temperature (18℃to 28 ℃, preferably 20℃to 25 ℃) for about 4 hours, and TLC detection was performed. Purification by prep plate (MeOH/dcm=1:20) was performed directly after the reaction, and tacrolimus derivative (51 mg, 47% yield) was finally obtained.

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

This example provides tacrolimus with a group that can bind to enzymes.

EXAMPLE 2 coupling of Tacrolimus derivatives with G6PDH molecules

1. Coupling methods of the present application

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

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

g6pdh solution: g6PDH (mutant of the present application or prior art mutant) was dissolved in PB 100mmol, naCl 100mmol, pH=8.0, 5mg/mL enzyme;

3.2 ml of glucose 6 phosphate dehydrogenase mutant solution, 7.5ml of PB solution and 0.5ml of tacrolimus derivative solution;

4. the above mixed solution was thoroughly shaken at room temperature (18-28deg.C, preferably 20-25deg.C) for 2-3 hours, and desalted (desalted solution 100mM PB, 0.1% NaN) ₃ 1% nacl, ph=8.0), protein peaks were collected, and the resulting product, G6 PDH-tacrolimus conjugate.

2. Control coupling method

Preparation of G6 PDH-tacrolimus conjugate with reference to the procedure disclosed in CN108107200a example:

1. dissolving 20-100mg of tacrolimus in methanol, adding anhydrous sodium acetate, adding carboxymethyl hydroxylamine after dissolving uniformly, dissolving and mixing uniformly, heating under the protection of nitrogen for reaction overnight, then distilling under reduced pressure to obtain wax, adding dimethylformamide for dissolving, filtering to remove precipitate, and distilling under reduced pressure to remove solvent to obtain a product A;

2. dissolving 10-50mg of product A with 20-100mL of dimethylformamide, slowly dripping 50-150 mu L of carbodiimide (EDC) into the solution under stirring, and mixing for 60-150 minutes in a rotating way;

3. dissolving 10-50mg glucose dehydrogenase with the specification of 100-300KU in PBS buffer solution, and shaking uniformly;

4. slowly adding the tacrolimus solution in the step 2 into the tacrolimus solution in the step 3 under stirring, and stirring and reacting for 8-16 hours.

EXAMPLE 3 preparation of the kit

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

reagent R1 comprising:

HEPES buffer 50mM, pH 7.0

10mM glucose 6-phosphate

10mM beta-nicotinamide adenine dinucleotide

1 μg/ml tacrolimus antibody (commercially available antibody)

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

200mM Tris buffer, pH 8.0

1 μg/ml G6 PDH-tacrolimus conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

sample extract: the volume ratio of methanol to ethanol is 3:1, and 1% zinc sulfate;

calibration material: 20mM HEPES buffer, 0.0, 2.5, 5.0, 10.0, 20.0, 30.0ng/ml tacrolimus (or added as needed);

quality control product: 20mM HEPES buffer, 8.1ng/ml, 15.4ng/ml, 23.2ng/ml (or added as needed).

The reagent (optionally comprising quality control substances and calibrator) is assembled into the tacrolimus homogeneous enzyme immunoassay kit.

Test case

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

Completely mixing human whole blood samples, quality control products and calibration products, taking 200 mu l of samples into corresponding centrifuge tubes by using a liquid transfer device, immediately covering a cover after adding an equal volume of sample extract, vibrating on a vortex vibration instrument for at least 10 seconds to ensure that the samples are fully and uniformly mixed, centrifuging at a speed of 12000r/min for 5 minutes on a centrifuge, transferring each supernatant into a small tube, and covering the cover, wherein the samples can be used for detection.

TABLE 1 full automatic Biochemical instrument parameters

Model type	Hitachi 7180 parameter
		Analysis point	[Rate-A][10][25][34]
WAVE(SUB/MAIN)	[410][340]
		S.VIL.	[20]
S.R1	[150]
		S.R3	[50]
ABS.LIMIT:	[32000][ incremental increase ]]
		CALIB TYPE:	[Spline]
POINT:	[6]SPAN PONIT[6]
		Calibration material	0.0、2.5、5.0、10.0、20.0、30.0ng/ml
Sample of	Samples to be tested, e.g. plasma, serum, whole blood, urine, etc

Test example 1. Performance of the kit of the present application

1. Scaling absorbance

TABLE 2 calibration absorbance

2. Precision experiments

And (3) measuring high, medium and low quality control products and clinical samples by using the calibration curve established above.

TABLE 3 Total imprecision

3. Repeatability of

TABLE 4 cassette repeatability

4. Recovery of

TABLE 5 recovery

5. Tacrolimus detection kit linearity

TABLE 6 linearity

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6. Tacrolimus 37 ℃ reagent acceleration stability

The calibrated absorbance decreases by about 16% after 7 days of acceleration at 37℃for the reagent of the present application, and about 51% after 7 days of acceleration at 37℃for the control reagent.

TABLE 7 accelerated stability of reagents at 37℃

Detection example 2 antibody inhibition Rate

1. Principle of detection of antibody inhibition

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

2. Reaction system

TABLE 8 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-tacrolimus 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.

Compared with published mutation sites (A45C), the mutant has obviously improved antibody inhibition rate, can reach more than 45% (G426C: 45%; D375C: 57%), and can reach up to 58% (D306C). Whereas the inhibition rates of the previously published mutation sites (e.g.A45C, K55C) were 41% and 25%.

TABLE 9 antibody inhibition by different G6PDH mutants

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 tacrolimus is prepared into a kit, the reagent has obvious performance improvement in the aspects of the batch variation coefficient, linearity, specificity and the like.

Sequence listing

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 3

<211> 486

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (375)..(375)

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

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 4

<211> 486

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (426)..(426)

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

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims

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

the hapten is a tacrolimus derivative;

the tacrolimus derivative is represented by formula I:

the compound of the formula I,

wherein,,

m is 1;

the glucose 6-phosphate dehydrogenase mutant comprises one mutation as compared to the wild-type glucose 6-phosphate dehydrogenase selected from the group consisting of: d306C, D375C; and the glucose 6-phosphate dehydrogenase mutant is shown as SEQ ID No.2 or SEQ ID No. 3.