CN111239060B

CN111239060B - 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent

Info

Publication number: CN111239060B
Application number: CN201911365439.3A
Authority: CN
Inventors: 龚俊; 祁金祥; 雷志; 张启飞; 王贵利; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2019-12-26
Publication date: 2023-04-07
Anticipated expiration: 2039-12-26
Also published as: CN116859035A; CN116840462A; CN117030640A; CN116840468A; CN111487207B; CN111693473A; CN116148198A; CN116718761A; CN116626281A; CN116735512A; CN116124721A; CN111504921A; CN116773827A; CN112285038A; CN111487207A; CN116298330A; CN111650135A; CN111678874A; CN116699125A; CN116773795A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a theophylline 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 and 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 theophylline detection reagent

Technical Field

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

Background

Haptens, some small molecular substances (molecular weight less than 4000 Da), 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 has only immunoreactivity and 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 specific structure are called simple hapten.

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

Theophylline (Theo) belongs to the xanthine alkaloids. Theophylline is usually made into salts (such as aminophylline) with high water solubility for medicinal use, but theophylline is still dissociated in vivo to play a role.

Theophylline is a bronchial smooth muscle relaxant, and is an effective drug for treating respiratory diseases such as bronchial asthma, chronic bronchitis, emphysema and chronic obstructive pulmonary disease. It is recorded in the guidelines for diagnosis and treatment of chronic obstructive pulmonary disease and bronchial asthma. Recent researches show that theophylline also has the effects of tonifying heart, promoting urination, resisting inflammation, regulating immunity, expanding coronary artery, exciting central nervous system and the like.

Due to the wide application of the theophylline medicines, the adverse reaction cases in patients are increased correspondingly, the common adverse reactions comprise nausea, vomiting, stomach cold and tachycardia, and some patients have nervous system symptoms. According to the research, the adverse reaction occurrence condition of the patient after using the theophylline medicine has a direct relation with the blood concentration of the patient. For most patients, blood concentrations of theophylline between 10 and 20 μ g/ml are effective in relieving chronic asthma and other bronchospasm symptoms. The blood concentration of theophylline between 5 and 10 mug/ml can control the neonatal apnea attack without obvious side effects.

However, the following adverse reactions occurred at blood concentrations exceeding 20. Mu.g/ml: nausea, headache, diarrhea. At higher plasma concentrations, vomiting, gastrointestinal bleeding, seizures and arrhythmias can occur.

Because the therapeutic index of theophylline is narrow, and the individual difference of metabolic rate and clearance rate is large, adverse reaction or poor treatment effect can be caused. Therefore, monitoring the blood concentration of the patient and adjusting the dosage at any time are very important for reducing the incidence rate of adverse reactions of the patient and improving the treatment effect.

The currently known theophylline detection methods mainly comprise: the method comprises high performance liquid chromatography, latex enhanced immunoturbidimetry, chemiluminescence microparticle immunization, enzyme-linked immunosorbent assay and the like. However, these detection methods all have many defects, such as radioactive contamination of radioimmunoassay isotope, short validity period, inconvenient operation and the like, and the enzyme-linked immunosorbent assay is relatively complicated in operation, takes a long time and is not suitable for clinical use. Although the chemiluminescence has better sensitivity, the chemiluminescence needs matched special equipment, and the investment and use cost is higher, which is not beneficial to 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-theophylline) and a non-labeled antigen (theophylline) compete for binding with a quantitative antibody (theophylline antibody), when the more the antibody is bound 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 the theophylline in the liquid can be calculated.

Disclosure of Invention

In view of the need in the art, the present application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in preparing a theophylline assay kit.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In distinction from the previously published mutants of glucose-6-phosphate dehydrogenase of patent US006090567A (nutritional glucose-6-phosphate dehydrogenases), the glucose-6-phosphate dehydrogenase mutants of the present application comprise mutations selected from the group consisting of: D306C, G426C and D375C.

According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

According to some embodiments, there is provided a polynucleotide encoding a glucose-6-phosphate dehydrogenase mutant of the present application.

According to some embodiments, there is provided an expression vector comprising a polynucleotide of the present application.

According to some embodiments, there is provided a host cell comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate of a glucose-6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1:1 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 coupling to glucose-6-phosphate dehydrogenase, haptens (e.g., theophylline) that do not themselves bear a coupling group (e.g., a group that reacts with a thiol group) can be engineered to have a linker to covalently bind to the thiol group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.

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

Haptens are exemplified by, but not limited to: theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside, mycophenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, cotinine metabolite cotinine, uromorphine, urinary monohydroxyphenol derivatives, 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, thyroxine, and mixtures thereof 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 particular embodiment, the hapten is theophylline or a derivative thereof.

In particular embodiments, the hapten is a theophylline derivative bearing a sulfhydryl-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine. In a particular embodiment, the hapten is a theophylline derivative, as shown in formula I:

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 theophylline detection reagent.

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

-a first reagent comprising a substrate and a theophylline antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the present application;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0mg/L to 40mg/L theophylline; and

-optionally, a quality control comprising 10mM to 500mM buffer, 0mg/L to 40mg/L theophylline.

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

a first reagent comprising:

10mM to 500mM buffer solution,

0.5g/L to 20g/L of substrate,

0.1mg/L to 10mg/L of theophylline antibody,

10mM to 300mM NaCl,

0.1g/L to 5g/L stabilizer,

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

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

Conjugates according to the present application,

0.1g/L 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, preferably 7.2 or 7.0. In some specific embodiments, the concentration of the buffer is 100mM.

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 specific embodiments, the concentration of the stabilizer is 1.0g/L.

In some embodiments, the surfactant is selected from one or a combination of: brij35, tritiom X-100, tritiom X-405, tween20, tween30, tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20. In some specific embodiments, the concentration of surfactant is 1.0g/L.

In some embodiments, the preservative is selected from one or a combination of: azide, MIT, PC-300, thimerosal; the azide is selected from: sodium azide and lithium azide. In some specific embodiments, the concentration of the preservative is 1.0g/L.

In some embodiments, the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide. In some specific embodiments, the substrate concentration of the reaction catalyzed by the G6PDH enzyme is 15G/L.

In some specific embodiments, the concentration of the theophylline antibody is 5.7mg/L.

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

providing a glucose-6-phosphate dehydrogenase mutant;

providing theophylline or a derivative thereof;

the 6-phosphoglucose dehydrogenase mutant and the theophylline or the derivative thereof are mixed according to a molar ratio of 1:1 coupling.

In some specific embodiments, the method of making the conjugate comprises the steps of:

1) Providing a theophylline derivative in an aprotic solvent;

2) Providing a glucose-6-phosphate dehydrogenase mutant in a buffer;

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

4) Optionally, the conjugate is purified, preferably desalted.

Wherein steps 1) and 2) are interchangeable.

In some specific embodiments, the aprotic solvent is selected from one or a combination of: acetonitrile, dimethylformamide, dimethyl sulfoxide.

In some specific embodiments, prior to step 3), the glucose-6-phosphate dehydrogenase mutant comprises one or more (preferably one) free thiol groups, especially a free thiol group present at position 306, 375 or 426.

Drawings

Figure 1. Theophylline structure diagram.

FIG. 2 shows the structure of theophylline derivatives.

FIG. 3A. G6PDH (wild-type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides of Leuconostoc.

FIG. 3B.G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

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

FIG. 3D.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

Example 1 Synthesis of theophylline derivatives

1. Synthesis of Compound 3

Theophylline (1.0g, 5.55mmol) and K ₂ CO ₃ (1.53g, 11.10mmol) was dissolved in 50mL of DMF, and Compound 2 (0.52g, 5.55mmol) was added thereto at room temperature (20-25 ℃ C.) and stirred for 16h. The solvent was removed under reduced pressure, the mixture was dissolved in 50mL of water, extracted three times with ethyl acetate (40 mL), washed three times with 50mL of saturated brine, and dried over anhydrous Na ₂ SO ₄ And (5) drying. The solvent was removed under reduced pressure and purified by column chromatography to give compound 3 (1.0 g, yield 76%).

2. Synthesis of theophylline derivatives

Compound 3 (100mg, 0.72mmol) and compound 4 (126mg, 0.72mmol) were dissolved in 5mL of DCM, triethylamine (218mg, 2.16mmol) was added dropwise thereto, HATU (328mg, 0.86mmol) was added, and the mixture was stirred at room temperature for 5 hours. Water (30 mL) was added to the reaction system, extraction was performed with DCM, and the organic phase was washed with saturated brine and anhydrous Na ₂ SO ₄ The mixture is dried and then is dried,the solvent was removed under reduced pressure to give the theophylline derivative (125 mg, yield 48%) as a colorless oil.

The product structure was verified by conventional methods. This example allows theophylline to have a group that can bind to an enzyme.

Example 2 coupling of theophylline derivatives to G6PDH molecules

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

1. Solution preparation:

theophylline derivative solution: theophylline derivative prepared in example 110 mg/ml in DMF;

g6PDH solution: g6PDH (mutant of the present application or prior art mutant) was soluble in PB 100mmol, naCl 100mmol, pH =8.0;

coupling solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH =7.2;

desalting solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH =7.2.

2. Coupling operation: 0.6ml of G6PDH solution, 4.18ml of the coupling solution and 0.22ml of the theophylline derivative solution were reacted at room temperature (20 to 25 ℃) for 4 hours.

3. And (3) oscillating the reaction system at room temperature for 4h, eluting with the desalting solution by using a desalting column, and collecting a protein peak to obtain a product, namely the G6 PDH-theophylline conjugate.

Example 3 preparation of the kit

A kit for detecting theophylline was prepared comprising:

a reagent R1 comprising:

100mM PB buffer, pH 7.2

15mM glucose 6-phosphate

15mM beta-nicotinamide adenine dinucleotide

5.7mg/L theophylline antibody (commercially available antibody, without particular limitation)

150mM NaCl

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

a reagent R2 comprising:

100mM PB buffer, pH 7.2

0.1mg/L G6 PDH-theophylline conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration products: 100mM PB buffer, pH 7.2, and 0, 2.4, 5.0, 10, 20, 40mg/L theophylline (or added as needed);

quality control product: 100mM PB buffer, pH 7.2, and 5.0, 15.0, 25mg/L theophylline (or added as needed).

Example of detection

Reaction time: 10min, wherein the incubation time is 4.7min, after 1min of incubation after adding the reagent R2, measuring the read absorbance A1, after 1min of incubation, measuring the read absorbance A2, calculating Δ a = (A2-A1)/min. The theophylline content of the sample was calculated from the calibration curve:

theophylline = sample tube absorbance calibrator concentration/calibrator absorbance.

The theophylline assay kit prepared in example 3 was subjected to performance testing, and the main testing properties were gross inaccuracy, reproducibility, recovery, linearity, and 37 ℃ accelerated stability.

TABLE 1 parameters of fully automatic biochemical analyzer

Detection example 1 Theine detection kit calibration Absorbance

TABLE 2 Theine assay kit calibration absorbance

Note: the mutation site of the prior art mutant with the A45C code corresponds to the 46 th position in FIG. 3A.

Detection example 2 Total inaccuracy of theophylline detection kit

TABLE 3 Total inaccuracy

Detection example 3 Theine detection kit repeatability

TABLE 4 repeatability

Detection example 4 Theine detection kit recovery

TABLE 5 recovery

Detection example 5 theophylline detection kit Linearity

TABLE 6 linearity

Test example 6.37 ℃ accelerated stability

TABLE 7.37 ℃ accelerated stability

After the application reagent is accelerated for 7 days at 37 ℃, the calibration absorbance is reduced by about 15 percent, and after the contrast reagent is accelerated for 7 days at 37 ℃, the calibration absorbance is about 95 percent.

Test example 7 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-theophylline 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

TABLE 9 antibody inhibition Rate testing of on-machine parameters

Detecting machine type	Yapei C16000
		analysis/time/Point	Speed/10 min/25-33
R1/S	120:20
		Wavelength (auxiliary/main)	405/340
Type of reaction	Incremental increase

3. As a result:

and (3) comparing the absorbance values of the G6 PDH-theophylline conjugate when the antibody is added with the antibody and when the antibody is not added, respectively detecting the absorbance values of the G6 PDH-theophylline conjugate, and thus obtaining the inhibition condition of the antibody on G6 PDH.

Antibody inhibition rate = [1- (change in absorbance of G6 PDH-theophylline with antibody/change in absorbance of G6 PDH-theophylline without antibody) ] × 100%.

Compared with published mutation sites, the mutant of the application has obviously improved antibody inhibition rate which can reach more than 30 percent and can reach as high as 55 percent. Whereas the inhibition rate of the mutation sites (such as A45C and K55C) commonly used before is only about 40% at most, and even lower.

TABLE 10 antibody inhibition of different G6PDH mutants

While not being bound to a particular theory, it may be partially explained as: compared with the G6PDH mutant in the prior art, the mutant (D306C, D375C, G426C) of the enzyme of the application has a mutation site (i.e., a site for introducing a free sulfhydryl) at a position for coupling with a 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 theophylline is prepared into the kit, the reagent has obvious performance improvement in the aspects of repeatability, total inaccuracy, linearity, stability and the like.

Sequence listing

<110> Beijing Jiuqiang Biotechnology Ltd

<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent

<130> 390269CG

<150> 201910017764.4

<151> 2019-01-09

<150> 201910423122.4

<151> 2019-05-21

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 486

<212> PRT

<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 2

<211> 486

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (306)..(306)

<223> G6PDH mutant, D at position 306 was replaced with C compared to wild type

<400> 2

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

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 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

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 formed by coupling a 6-phosphoglucose dehydrogenase mutant and a theophylline derivative;

the theophylline derivative is represented by formula I:

the glucose-6-phosphate dehydrogenase mutant comprises the mutation D306C compared to a wild-type glucose-6-phosphate dehydrogenase;

the 6-phosphoglucose dehydrogenase mutant is shown as SEQ ID No. 2.

2. The use of the conjugate of claim 1 for the preparation of a homogeneous enzyme immunoassay detection reagent:

the detection reagent is theophylline.