CN111693473A

CN111693473A - 6-phosphoglucose dehydrogenase mutant and application thereof in preparing rapamycin detection reagent

Info

Publication number: CN111693473A
Application number: CN202010016535.3A
Authority: CN
Inventors: 王贵利; 张启飞; 李垚艳; 龚俊; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2020-01-08
Publication date: 2020-09-22
Anticipated expiration: 2040-01-08
Also published as: CN117054643A; CN116718764A; CN116124721A; CN111650135A; CN111537451A; CN112285037A; CN116338215A; CN116840467A; CN111504920A; CN116718761A; CN116626281A; CN116735512A; CN116148198A; CN117030640A; CN112285038A; CN111537452B; CN116359146A; CN116699122A; CN116773795A; CN111487207A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a rapamycin detection reagent. Specifically, the glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations selected from the group consisting of: D306C, D375C, G426C. The detection kit prepared by using the glucose-6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time and accurate quantification, and is suitable for high-throughput detection.

Description

6-phosphoglucose dehydrogenase mutant and application thereof in preparing rapamycin detection reagent

Priority of application No. 201910017764.4 filed on 1/9/2019 and 201910423122.4 "mutant glucose-6-phosphate dehydrogenase and use thereof in the preparation of test agents" filed on 5/21/2019, which are incorporated herein by reference.

Technical Field

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

Background

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

The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and will stimulate the production of corresponding antibodies in animals.

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

Rapamycin (Rapamune) has the structural formula shown below:

rapamycin (also known as sirolimus, siro) is a hydrophobic macrocyclic triene lactone synthesized by actinomycetes, a member of the lipophilic family of molecules. Which carries a lactone ring substituted at the 12-, 14-or 16-position with hydroxy, methyl, or ethyl. When the rapamycin is used together with the cyclosporine and the glucocorticoid, the incidence rate of acute rejection reaction of a kidney transplant receptor can be reduced. The reason is that rapamycin binds to tacrolimus binding protein-12, and inhibits the proliferation of T lymphocytes.

Rapamycin is distributed in human erythrocytes, metabolized by the liver, and cleared by feces and bile. Side effects of rapamycin are headache, nausea, dizziness, epistaxis, joint pain; laboratory examination finds the following indicators abnormal: thrombocytopenia, leukopenia, hypertriglyceridemia, hypercholesterolemia, etc. The above side effects are dose-dependent and reversible.

For the reasons, rapamycin blood concentration monitoring needs to be carried out in time in the treatment process, and the method is an effective mode for assisting clinical treatment, improving treatment effect and reducing toxicity risk.

The currently known rapamycin detection methods are mainly: high Performance Liquid Chromatography (HPLC), luminescence immunity, enzyme-linked immunosorbent assay (ELISA), etc. The high performance liquid chromatography can separate the drug, metabolite and endogenous substance, has the characteristic of strong specificity, is a gold standard for detecting MTX plasma concentration, but the method needs a complex pretreatment process and longer determination time, and is not suitable for the rapid detection of large samples. 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-rapamycin) and a non-labeled antigen (rapamycin) compete for being combined with a quantitative antibody (rapamycin antibody), when the antibody is more combined with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH is generated by catalyzing a substrate NAD +, and the change of absorbance of NADH is detected at the wavelength of 340nm, so that the content of the rapamycin in the liquid can be calculated.

The existing homogeneous enzyme immunoassay method depends on activating a reactive group carried by a small molecule drug and then reacting with 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 need in the art, the present application provides a novel mutant of glucose-6-phosphate dehydrogenase and its use in the preparation of a rapamycin detection kit.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the previously published mutant of glucose-6-phosphate dehydrogenase of patent US006090567A (halogenated immunological systems using mutant glucose-6-phosphate dehydrogenes), the glucose-6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: D306C, D375C, G426C.

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: 1 is coupled.

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., rapamycin) that do not themselves bear a coupling group (e.g., a group reactive with a thiol group) can be engineered to bear a linker for covalent attachment to the thiol group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.

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

Haptens are exemplified by, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin, digoxigenin), mycophenolic acid, rapamycin, cyclosporine A, methotrexate, amiodarone, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites cotinine of uretonidine, morphine, derivatives of uromonohydric phenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free 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 prostaglandins, plasma 6-keto prostaglandin F1 α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane a2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

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

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

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

wherein the content of the first and second substances,

SIRO stands for

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

In a particular embodiment, the rapamycin derivative is of formula II:

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

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

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

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

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

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

-a first reagent comprising a substrate, a buffer and a rapamycin 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, rapamycin at a known concentration; and

-optionally, a quality control comprising a buffer of 10mM to 500mM, a known concentration of rapamycin.

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

0.01 to 10. mu.g/ml of a rapamycin antibody, 0.1 to 5g/L of a stabilizer,

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

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

0.01. 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: TAPS, tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 50 to 300 mM; the pH of the buffer is 7 to 8.4.

In some embodiments, the stabilizing agent is selected from one or a combination of: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; bovine serum albumin is preferred.

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

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

In some embodiments, the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.

In some specific embodiments, the rapamycin antibody is derived from: rabbit, mouse, rat, goat, sheep, cat, guinea pig, dog, primate, cow, horse, camelid, avian, human.

In some specific embodiments, the rapamycin 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 rapamycin derivative according to the present application, in particular providing a rapamycin derivative according to the present application 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) contacting said rapamycin derivative and said glucose-6-phosphate dehydrogenase mutant at a molar ratio n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) at 18 ℃ to 28 ℃ to couple said rapamycin derivative and said glucose-6-phosphate dehydrogenase mutant to provide said conjugate;

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

In some embodiments, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein n is 1 to 500, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the foregoing values; preferably n is 20 to 60, for example 50.

In some specific embodiments, steps 1) and 2) can be interchanged or in parallel.

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

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

Drawings

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

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

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

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

Detailed Description

Examples

EXAMPLE 1 Synthesis of rapamycin derivatives

To a round bottom flask was added 10mL of dried DCM, to which was added Compound 1(200mg, 1.18mmol), DIPEA (183mg, 1.42mmol), cooled to 0 deg.C, slowly added Compound 2(244mg, 1.18mmol) under nitrogen, the reaction temperature was raised to room temperature (18-28 deg.C, preferably 20 to 25 deg.C), stirred further, checked by TLC, about 4 hours after the reaction was complete and used in the next step without treatment.

Adding rapamycin (1.08g, 1.18mmol) into the reaction system, adding DMAP (432mg, 3.54mmol), stirring at room temperature, detecting by TLC, removing the solvent under reduced pressure after the reaction is finished, and purifying by column chromatography to obtain rapamycin derivative (480mg) with the yield of 38%.

The structure of the product was confirmed by a conventional method. This example allows rapamycin to carry a group that can bind to an enzyme.

Example 2 coupling of rapamycin derivatives to G6PDH molecules

First, the coupling method of the present application

The G6 PDH-rapamycin 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 rapamycin derivative molecule is covalently bound to a thiol on a G6PDH molecule.

1. Dissolving rapamycin derivative in N, N-dimethylformamide (10 mg/ml);

2. mu. l G6PDH (mutant of the present application or mutant of the prior art) solution (6.4mg/ml, 0.2M phosphate buffer, pH 8.0) was added to 750. mu.l buffer solution (0.05M Na)₂HPO₄、150mM NaCl、10mM EDTA、0.1％NaN₃pH 7.2);

3. then 50. mu.l of a solution of rapamycin derivative in N, N-dimethylformamide was added thereto;

4. fully shaking the mixed solution at room temperature (18-28 ℃) for 2-3 hours;

5. molecular sieve chromatography gave G6 PDH-rapamycin conjugate (concentration 0.1mg/mL-2.0 mg/mL).

Two, non-directed control coupling method (relying on activation of rapamycin self-carrying groups)

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

2. dissolving a certain amount of rapamycin, 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 rapamycin solution into dissolved G6PDH, and stirring for dissolving;

4. stirring at 2-8 deg.C for dissolving overnight;

5. after purification, the resulting glucose dehydrogenase-rapamycin conjugate was stored at 2-8 ℃.

Example 3 preparation of the kit

Preparing the following kit for detecting rapamycin, comprising:

1. preparation of the first reagent:

2. preparation of the second reagent:

3. quality control product and calibrator:

the quality control substance is rapamycin pure product and is obtained by diluting a buffer solution, and the concentration of the rapamycin pure product is 4-6ng/ml, 8-12ng/ml and 22-28ng/ml respectively.

The calibration substance is rapamycin pure product and is obtained by diluting a buffer solution, and the concentrations of the calibration substance are 0ng/ml, 3ng/ml, 6ng/ml, 12ng/ml, 24ng/ml and 36ng/ml respectively.

4. Assembling a kit:

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

Example of detection

In a homogeneous reaction system, rapamycin and G6 PDH-rapamycin conjugate in a sample compete for binding to an anti-rapamycin antibody site simultaneously, the more rapamycin in the sample is free, the more antibody sites that compete for binding, the less antibody is bound to the enzyme conjugate, and the enzyme conjugate that is not bound to the antibody catalyzes β -nicotinamide adenine dinucleotide oxidation (NAD) due to decreased enzyme activity after the antibody binds to the conjugate⁺) Converting into β -nicotinamide adenine dinucleotide reduced type (NADH), wherein the concentration of rapamycin in the sample is in direct proportion to the generation amount of NADH, and obtaining the concentration of rapamycin in the sample through the change of absorbance.

TABLE 1 parameters of fully automatic biochemical analyzer

Test example 1 Performance of the kit of the present application

1. Calibration of absorbance

TABLE 2 calibrated absorbances

2. Precision experiment

And measuring the high, medium and low quality control products by using the calibration curve established above.

TABLE 3 precision (D306C mutant)

3. Repeatability of

TABLE 4 repeatability

4. Recovering

TABLE 5 recovery

5. Linearity

TABLE 6 linearity

6. Stability of

TABLE 7.37 ℃ accelerated stability

Detection example 2 inhibition ratio of antibody in conjugate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-rapamycin 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 in which the antibody is added and an experimental group in which the antibody is not added is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.

2. Reaction system

TABLE 8 preparation of reagents for measuring antibody inhibition

3. Results

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

Compared with the conjugate prepared from the published mutation sites (A45C and K55C), the enzyme mutant has obviously improved antibody inhibition rate which can reach more than 50 percent (G426C: 50 percent; D375C: 51 percent) and can reach up to 60.4 percent (D306C). Whereas the inhibition of previously published mutation sites (e.g. a45C, K55C) is 38.6% to 45%.

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

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

Sequence listing

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210>4

<211>486

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>VARIANT

<222>(426)..(426)

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

<400>5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275280 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

435440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims

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

the hapten is rapamycin or a derivative thereof;

preferably, the rapamycin derivative is of formula I:

wherein the content of the first and second substances,

SIRO in formula I

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

preferably, the rapamycin derivative is of formula II:

2. the conjugate of claim 1, wherein:

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

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

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

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

the detection reagent is a rapamycin detection reagent;

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

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

the detection device is a rapamycin-directed detection device;

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

6. A rapamycin detection kit comprising:

a first agent comprising a substrate, an anti-rapamycin antibody, a buffer;

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

optionally, a calibrator comprising 10mM to 500mM buffer, 0ng/ml to 40ng/ml rapamycin; and

optionally, a quality control comprising 10mM to 500mM buffer, 4ng/ml to 30ng/ml rapamycin.

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

a first reagent comprising:

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

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

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

0.01 to 10. mu.g/ml, preferably 0.1 to 1. mu.g/ml, of an anti-rapamycin antibody,

0.1 to 5g/L, preferably 0.5 to 2g/L, of a stabilizer,

0.1 to 5g/L, preferably 0.5 to 2g/L, of a surfactant,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L preservative;

a second reagent comprising:

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

The conjugate of claim 1 or 2, in an amount of 0.01 to 10. mu.g/ml, preferably 0.1 to 1. mu.g/ml,

0.1 to 5g/L, preferably 0.5 to 2g/L, of a stabilizer,

0.1 to 5g/L, preferably 0.5 to 2g/L, of a surfactant,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L preservative;

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

the pH of the buffer is 7 to 8.4;

the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably bovine serum albumin;

the surfactant is selected from: triton X-100, TritonX-405, Tween80, Tween20, Brij35, Brij23, or a combination thereof, preferably Tween 20;

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

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

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

a first reagent comprising:

a second reagent comprising:

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

1) providing rapamycin or a derivative thereof;

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

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

the rapamycin derivative is represented by formula I:

wherein the content of the first and second substances,

SIRO stands for

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

preferably, the rapamycin derivative is of formula II:

10. a method of preparing a conjugate according to claim 9, comprising the steps of:

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

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

3) contacting the glucose-6-phosphate dehydrogenase mutant and the rapamycin derivative at 18 ℃ to 28 ℃, preferably 20 ℃ to 25 ℃, for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that conjugation of the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant occurs, resulting in the conjugate;

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

step 1) and step 2) can be interchanged or in parallel;

the buffer is selected from: phosphate buffer, Tris buffer, Hepes buffer, PBS buffer, TAPSO buffer,

the pH of the buffer 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), said glucose-6-phosphate dehydrogenase mutant comprises a free thiol group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375, or 426;

preferably, the rapamycin derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1, n is 1 to 500, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500;

more preferably, n is 30 to 60; most preferably, n is 50.