Preparation method of conjugate

Abstract

This application relates to a method for preparing a conjugate. Specifically, the 6-glucose phosphate dehydrogenase mutant of this application comprises one or a combination of the following mutations compared to wild-type 6-glucose phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the 6-glucose phosphate dehydrogenase mutant of this application has strong specificity, high sensitivity, convenient operation, short detection time, and accurate quantitative determination, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of the patent application "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Theophylline Detection Reagent" filed on December 26, 2019, with application number: 2019113654393. Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a multi-site mutated enzyme 6-phosphate glucose dehydrogenase (G6PDH for short) and its application in a theophylline detection kit. Background Art

[0003] Haptens are small molecules (molecular weight less than 4000 Da) that cannot induce an immune response on their own, meaning they are not immunogenic. However, when cross-linked or bound to a macromolecular protein or a non-antigenic carrier such as polylysine, they can become immunogenic and induce an immune response. These small molecules can bind to effector products and become antigenic, but are only immunoreactive and not immunogenic, and are also called incomplete antigens.

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate the production of antibodies in humans or animals on its own. It is only immunoreactive and not immunogenic, and is also called an incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically combined with a certain protein molecule (carrier), it will acquire new immunogenicity and can stimulate animals to produce corresponding antibodies. Once a hapten binds to a protein, it forms an antigenic cluster of that protein. Some substances with smaller molecular weight than ordinary haptens but with specific chemically active groups (such as penicillin and sulfonamides) are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites for sandwich assays, so the double antibody sandwich assay cannot be used. Instead, a competitive assay is often used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen for binding to the solid-phase antibody. The greater the amount of antigen in the specimen, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This assay is often used for ELISA assays of small molecule hormones, drugs, etc.

[0006] Theophyline (Theo) belongs to the xanthine alkaloid family. Theophylline is usually prepared into highly water-soluble salts (such as aminophylline) for medicinal use, but it still dissociates into theophylline in the body to exert its effects.

[0007] Theophylline, a bronchial smooth muscle relaxant, is an effective treatment for respiratory diseases such as bronchial asthma, chronic bronchitis, emphysema, and chronic obstructive pulmonary disease. It is included in the "Guidelines for the Diagnosis and Treatment of Chronic Obstructive Pulmonary Disease" and the "Guidelines for the Prevention and Treatment of Bronchial Asthma." Recent studies have shown that theophylline also has cardiotonic, diuretic, anti-inflammatory, immunomodulatory, coronary artery dilation, and central nervous system stimulation effects.

[0008] Due to the widespread use of theophylline drugs, the number of adverse reactions among patients has also increased accordingly. The more common adverse reactions include nausea, vomiting, stomach cold, tachycardia, and some patients may also experience neurological symptoms. According to studies, the adverse reactions that occur after patients use theophylline drugs are directly related to the patient's blood drug concentration. For most patients, a theophylline blood concentration between 10 and 20 μg / ml can effectively relieve chronic asthma and other bronchospasm symptoms. A theophylline blood concentration between 5 and 10 μg / ml can control neonatal apnea attacks without obvious side effects.

[0009] However, adverse reactions such as nausea, headache, and diarrhea may occur when the blood concentration exceeds 20 μg / ml. At higher blood concentrations, vomiting, gastrointestinal bleeding, seizures, and cardiac arrhythmias may occur.

[0010] Because theophylline has a narrow therapeutic index and significant inter-individual variability in its metabolism and clearance rates, adverse reactions or poor therapeutic efficacy can occur. Therefore, monitoring the patient's blood drug concentration and adjusting the dosage at any time are crucial to reducing the incidence of adverse reactions and improving therapeutic efficacy.

[0011] Currently known theophylline detection methods mainly include: high performance liquid chromatography, latex enhanced immunoturbidimetry, chemiluminescent microparticles immunoassay, enzyme-linked immunosorbent assay (ELISA), etc. However, these detection methods all have more defects. For example, radioimmunoassay isotopes have many drawbacks such as radioactive contamination, a shorter shelf life, and inconvenient operation. The ELISA operation is more complicated and time-consuming, making it unsuitable for clinical use. Although chemiluminescence has better sensitivity, it requires supporting special equipment, and the higher cost of use is unfavorable for promotion. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex enhanced immunoturbidimetry are mainly used for detection.

[0012] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-theophylline) and unlabeled antigen (theophylline) compete with a quantitative antibody (theophylline antibody) for binding. The more the antibody binds to the unlabeled antigen, the more activity the enzyme-labeled antigen releases, and the more NADH the enzyme catalyzes from the substrate NAD+. By detecting the absorbance change of NADH at a wavelength of 340nm, the content of theophylline in the liquid can be inferred. Summary of the Invention

[0013] In view of the needs in the art, the present application provides a novel 6-phosphate glucose dehydrogenase mutant and its use in preparing a theophylline detection kit.

[0014] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided. Different from the 6-phosphate glucose dehydrogenase mutant disclosed in the previously published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphate glucose dehydrogenase mutant of the present application comprises mutations selected from the group consisting of: D306C, G426C, and D375C.

[0015] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided, wherein the 6-phosphate glucose 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.

[0016] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0017] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0018] According to some embodiments, a host cell is provided, comprising the expression vector of the present application. The host cell can be a prokaryotic cell (such as a bacterium) or a eukaryotic cell (such as a yeast).

[0019] According to some embodiments, a conjugate is provided, which is formed by coupling the 6-phosphate glucose dehydrogenase mutant of the present application with a hapten at a molar ratio of 1:1.

[0020] In some specific embodiments, the molecular weight of the hapten is 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, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210 000, 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.

[0021] According to the present application, skilled artisans will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as theophylline) that do not themselves carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker to facilitate covalent binding with the sulfhydryl group. Therefore, in the present application, a hapten derivative refers to a hapten that has been modified to carry a sulfhydryl reactive group.

[0022] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0023] Haptens include, but are not limited to, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides, phenolic acid, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, trans-triiodothyronine, free thyroxine, free triiodothyronine gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0024] In specific embodiments, the hapten is theophylline or a derivative thereof.

[0025] In a specific embodiment, the hapten is a theophylline derivative having a sulfhydryl reactive group, such as imide, bromoacetyl, vinyl sulfone or aziridine. In a specific embodiment, the hapten is a theophylline derivative, as shown in Formula I:

[0026]

[0027] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0028] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a theophylline detection reagent.

[0029] According to some embodiments, there is provided use of the conjugate of the present application in preparing a theophylline detection reagent.

[0030] In a specific embodiment, the detection reagent is selected from: enzyme-linked immunosorbent assay detection reagent, chemiluminescence immunoassay detection reagent, homogeneous enzyme immunoassay detection reagent, latex-enhanced immunoturbidimetric detection reagent.

[0031] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0032] According to some embodiments, a theophylline detection kit is provided, comprising:

[0033] - a first reagent, comprising a substrate and a theophylline antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0034] - a second reagent, which comprises the conjugate of the present application;

[0035] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 mg / L to 40 mg / L theophylline; and

[0036] -Optionally, a quality control, comprising 10 mM to 500 mM buffer, 0 mg / L to 40 mg / L theophylline.

[0037] According to one embodiment, a theophylline detection kit is provided, comprising:

[0038] The first reagent comprises:

[0039] 10mM to 500mM buffer,

[0040] 0.5g / L to 20g / L substrate,

[0041] 0.1mg / L to 10mg / L theophylline antibodies,

[0042] 10mM to 300mM NaCl,

[0043] 0.1g / L to 5g / L stabilizer,

[0044] 0.1g / L to 5g / L surfactant,

[0045] 0.1g / L to 5g / L preservatives;

[0046] A second reagent comprising:

[0047] 10mM to 500mM buffer,

[0048] According to the conjugate of this application,

[0049] 0.1g / L to 5g / L stabilizer,

[0050] 0.1g / L to 5g / L surfactant,

[0051] 0.1g / L to 5g / L preservatives.

[0052] In some embodiments, the buffer is selected from one or a combination of the following: tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, and tris(hydroxymethyl)methane buffer; preferably, phosphate buffer; the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 100 mM; 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 100 mM.

[0053] 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; preferably bovine serum albumin. In some specific embodiments, the concentration of the stabilizer is 1.0 g / L.

[0054] In some embodiments, the surfactant is selected from one or a combination of the following: Brij35, Tritiom X-100, Tritiom X-405, Tween 20, Tween 30, Tween 80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20. In some specific embodiments, the concentration of the surfactant is 1.0 g / L.

[0055] In some embodiments, the preservative is selected from one or a combination of the following: 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 1.0 g / L.

[0056] In some embodiments, the substrate comprises: 6-phosphoglucose, β-nicotinamide adenine dinucleotide. In some specific embodiments, the substrate concentration of the G6PDH enzyme-catalyzed reaction is 15 g / L.

[0057] In some specific embodiments, the concentration of theophylline antibody is 5.7 mg / L.

[0058] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0059] Providing a 6-phosphate glucose dehydrogenase mutant;

[0060] providing theophylline or its derivatives;

[0061] The 6-phosphate glucose dehydrogenase mutant is coupled with the theophylline or its derivative in a molar ratio of 1:1.

[0062] In some specific embodiments, the method for preparing the conjugate comprises the steps of:

[0063] 1) providing a theophylline derivative in an aprotic solvent;

[0064] 2) providing the 6-phosphate glucose dehydrogenase mutant in a buffer;

[0065] 3) contacting the theophylline derivative and the 6-phosphate glucose dehydrogenase mutant at 18° C. to 28° C. for 1 to 4 hours, preferably 2 to 3 hours, to allow the theophylline derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0066] 4) Optionally, the conjugate is purified, preferably desalted.

[0067] Among them, steps 1) and 2) are interchangeable.

[0068] In some specific embodiments, the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide.

[0069] In some specific embodiments, before step 3), the 6-phosphate glucose dehydrogenase mutant contains one or more (preferably one) free thiol groups, especially a free thiol group at position 306, 375 or 426. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 .Structure diagram of theophylline.

[0071] Figure 2 .Structure diagram of theophylline derivatives.

[0072] Figure 3A .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0073] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0074] Figure 3C .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0075] Figure 3D .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0076] Example

[0077] Example 1. Synthesis of theophylline derivatives

[0078]

[0079]

[0080] 1. Synthesis of Compound 3

[0081] Theophylline (1.0 g, 5.55 mmol) and K2CO3 (1.53 g, 11.10 mmol) were dissolved in 50 mL of DMF, and compound 2 (0.52 g, 5.55 mmol) was added at room temperature (20-25°C) and stirred for 16 h. The solvent was removed under reduced pressure, and the mixture was dissolved in 50 mL of water, extracted three times with ethyl acetate (40 mL), washed three times with 50 mL of saturated brine, and dried over anhydrous Na2SO4. The solvent was removed under reduced pressure, and the product was purified by chromatography to obtain compound 3 (1.0 g, 76% yield).

[0082] 2. Synthesis of theophylline derivatives

[0083] Compound 3 (100 mg, 0.72 mmol) and compound 4 (126 mg, 0.72 mmol) were dissolved in 5 mL of DCM, and triethylamine (218 mg, 2.16 mmol) was added dropwise. HATU (328 mg, 0.86 mmol) was also added, and the mixture was stirred at room temperature for 5 h. Water (30 mL) was added to the reaction system, and the mixture was extracted with DCM. The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure to obtain a colorless oily theophylline derivative (125 mg, 48% yield).

[0084] The product structure was verified by conventional methods. In this embodiment, theophylline is provided with a group that can bind to an enzyme.

[0085] Example 2. Coupling of theophylline derivatives with G6PDH molecules

[0086] According to the G6PDH-theophylline conjugate of the present application, the conjugation is carried out in the following manner: the sulfhydryl reactive group (such as but not limited to maleimide group) on the theophylline derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0087] 1. Solution preparation:

[0088] Theophylline derivative solution: 10 mg / ml of theophylline derivative prepared in Example 1 was dissolved in DMF;

[0089] G6PDH solution: G6PDH (mutant of the present application or mutant of the prior art) was dissolved in 100 mmol PB, 100 mmol NaCl, pH = 8.0;

[0090] Coupling solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2;

[0091] Desalting solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2.

[0092] 2. Coupling operation: 0.6 ml of G6PDH solution, 4.18 ml of coupling solution and 0.22 ml of theophylline derivative solution were reacted at room temperature (20 to 25°C) for 4 hours.

[0093] 3. After the reaction system was shaken at room temperature for 4 hours, it was eluted using a desalting column with the desalting solution, and the protein peak was collected. The obtained product was G6PDH-theophylline conjugate.

[0094] Example 3. Preparation of kit

[0095] The following kit for detecting theophylline was prepared, comprising:

[0096] Reagent R1, containing:

[0097] 100 mM PB buffer, pH 7.2

[0098] 15 mM glucose 6-phosphate

[0099] 15 mM β-nicotinamide adenine dinucleotide

[0100] 5.7mg / L theophylline antibody (commercially available antibody, no special restrictions)

[0101] 150mM NaCl

[0102] 1g / L bovine serum albumin

[0103] 1g / L Tween20

[0104] 1g / L sodium azide;

[0105] Reagent R2, including:

[0106] 100 mM PB buffer, pH 7.2

[0107] 0.1mg / L G6PDH-theophylline conjugate

[0108] 1g / L bovine serum albumin

[0109] 1g / L Tween 20

[0110] 1g / L sodium azide;

[0111] Calibrators: 100 mM PB buffer, pH 7.2, and 0, 2.4, 5.0, 10, 20, 40 mg / L theophylline (or as needed);

[0112] Quality control: 100 mM PB buffer, pH 7.2, and 5.0, 15.0, and 25 mg / L theophylline (or add as needed).

[0113] Test example

[0114] Reaction time: 10 minutes, including an incubation time of 4.7 minutes. After adding reagent R2 and incubating for 1 minute, the absorbance A1 is measured and read. After another 1 minute of incubation, the absorbance A2 is measured and read. Calculate ΔA = (A2 - A1) / min. Calculate the theophylline content in the sample using the calibration curve:

[0115] Theophylline = sample tube absorbance * calibrator concentration / calibrator absorbance.

[0116] The theophylline detection kit prepared in Example 3 was subjected to performance testing. The main test performances were total imprecision, repeatability, recovery, linearity, and 37°C accelerated stability.

[0117] Table 1. Parameters of fully automatic biochemical analyzer

[0118] Detection model Abbott C16000 Analysis / Time / Point Speed / 10min / 25-33 points R1 / R2 / S 150:50:2 Wavelength (secondary / primary) 405 / 340 Reaction type Increment Calibration type Spine type Calibration points 6 Calibrator concentration 0 / 2.4 / 5.00 / 10.00 / 20.00 / 40.00

[0119] Test Example 1. Absorbance calibration of theophylline test kit

[0120] Table 2. Calibrated absorbance of theophylline detection kit

[0121]

[0122] Note: The mutant code-named A45C in the prior art has a mutation site corresponding to Figure 3A No. 46.

[0123] Test Example 2: Total Imprecision of the Theophylline Test Kit

[0124] Table 3. Total imprecision

[0125]

[0126] Test Example 3. Repeatability of theophylline test kit

[0127] Table 4. Repeatability

[0128]

[0129] Test Example 4. Theophylline Detection Kit Recovery

[0130] Table 5. Recycling

[0131]

[0132]

[0133] Test Example 5. Linearity of theophylline test kit

[0134] Table 6. Linearity

[0135]

[0136]

[0137] Test Example 6.37℃ Accelerated Stability

[0138] Table 7. Accelerated stability at 37°C

[0139]

[0140] After the reagent of the present application is accelerated at 37°C for 7 days, the standardized absorbance decreases by about 15%, while the control reagent has a standardized absorbance of about 95% after being accelerated at 37°C for 7 days.

[0141] Test Example 7. Antibody Inhibition Rate

[0142] 1. Principle of Antibody Inhibition Rate Detection

[0143] When the antibody binds to the G6PDH-theophylline conjugate, the G6PDH enzyme activity is affected due to steric hindrance, thereby reducing its efficiency in catalyzing the conversion of NAD to NADH. By detecting the change in NADH amount, the difference between the experimental groups with and without the antibody is compared. This difference is reflected in the antibody's ability to inhibit G6PDH.

[0144] 2. Reaction system:

[0145] Table 8. Preparation of antibody inhibition rate detection reagents

[0146]

[0147] Table 9. Antibody inhibition rate test parameters

[0148] Detection model Abbott C16000 Analysis / Time / Point Speed / 10min / 25-33 R1 / S 120:20 Wavelength (secondary / primary) 405 / 340 Reaction type Increment

[0149] 3. Results:

[0150] By comparing the absorbance values ​​of the G6PDH-theophylline conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0151] Antibody inhibition rate = [1-(absorbance change of G6PDH-theophylline in the presence of antibody / absorbance change of G6PDH-theophylline in the absence of antibody)] x 100%.

[0152] Compared to previously published mutation sites, the mutants in this application have significantly improved antibody inhibition rates, reaching over 30% and up to 55%. Previously commonly used mutation sites (such as A45C and K55C) have inhibition rates of only around 40% or even lower.

[0153] Table 10. Antibody inhibition rates of different G6PDH mutants

[0154]

[0155] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants in the prior art, the mutation site (i.e., the site where the free thiol group is introduced) in the enzyme mutants of the present application (D306C, D375C, G426C) is the location where coupling occurs with the hapten (such as a hormone, a small molecule drug, etc.). When the hapten binds to the hapten-specific antibody at this position, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme. At the same time, after the mutation is introduced, it cannot substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is very important, and it is necessary to take into account the activity of the G6PDH enzyme, the spatial folding of the coupled molecule, and the full exposure of the hapten epitope.

[0156] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the reagent kit, after the enzyme mutant is coupled with theophylline, has a significant improvement in repeatability, total imprecision, linearity, stability and other performance.

Claims

1. A method for preparing a conjugate, comprising the steps of: Providing a 6-phosphate glucose dehydrogenase mutant; providing theophylline derivatives; The 6-phosphate glucose dehydrogenase mutant is coupled with the theophylline derivative; The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No. 2; The theophylline derivative is shown in formula I: Formula I.

2. The method for preparing the conjugate according to claim 1, comprising the steps of: 1) Providing theophylline derivatives in an aprotic solvent; 2) providing the 6-phosphate glucose dehydrogenase mutant in a buffer; 3) contacting the theophylline derivative and the 6-phosphate glucose dehydrogenase mutant at 18° C. to 28° C. for 1 to 4 hours to allow the theophylline derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate; Steps 1) and 2) are interchangeable; The buffer is selected from: PBS, Tris, TAPS, TAPSO, The buffer solution has a pH of 6.0 to 8.0; The aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide; Before step 3), the 6-phosphate glucose dehydrogenase mutant has a free sulfhydryl group at position 306.

3. The method for preparing the conjugate according to claim 1, wherein step 3) comprises: The theophylline derivative and the 6-phosphate glucose dehydrogenase mutant are contacted at 18° C. to 28° C. for 2 to 3 hours, so that the theophylline derivative and the 6-phosphate glucose dehydrogenase mutant are coupled to obtain the conjugate.

4. The method for preparing the conjugate according to claim 1, further comprising, after step 3), step 4) purifying the conjugate.

5. The method for preparing the conjugate according to claim 4, wherein: The conjugate was purified by desalting.

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