GLUCOSE-6-PHOSPHATE DEHYDROGENASE MUTANT AND USE THEREOF IN PREPARING DETECTION REAGENT

Abstract

Disclosed is a glucose-6-phosphate dehydrogenase mutant and a use thereof in preparing a detection reagent. Compared with a wild-type glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant contains a combination of the following mutations: 56C, 306C, and 454C. A detection kit prepared by using the glucose-6-phosphate dehydrogenase has strong specificity, high sensitivity, convenient operation, a short detection time, accurate quantification, and is suitable for high-throughput detection.

Claims

1. A glucose 6-phosphate dehydrogenase mutant, which comprises a combination of the following mutations compared to Leuconostoc pseudomesenteroides wild-type glucose 6-phosphate dehydrogenase: 56C, 306C and 454C.

2. A polynucleotide encoding the glucose 6-phosphate dehydrogenase mutant according to claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3, the host cell does not involve a cell capable of developing into an animal or plant.

5. A conjugate, which is obtained by conjugating the glucose 6-phosphate dehydrogenase mutant of claim 1 with a hapten at a molar ratio of 1:m; m is from 1 to 3.

6. A reagent comprising the conjugate of claim 5.

7. (canceled)

8. A detection kit comprising: a first reagent comprising a substrate, an anti-hapten antibody, and a buffer; a second reagent comprising the conjugate of claim 5 and a buffer; optionally, a calibrator comprising 10 mM to 500 mM buffer, a hapten with known concentration; and optionally, a quality control comprising 10 mM to 500 mM buffer and a hapten with known concentration.

9. The detection kit according to claim 8, comprising: a first reagent comprising: 10 mM to 500 mM buffer, 5 mM to 25 mM substrate, 0.01 m/L to 1 mg/L anti-hapten antibody, 10 mM to 300 mM NaCl, 0.1 g/L to 5 g/L stabilizer, 0.1 g/L to 5 g/L surfactant, 0.1 g/L to 5 g/L preservative; a second reagent comprising: 10 mM to 500 mM buffer, 0.01 m/L to 1 mg/L conjugate of claim 5, 0.1 g/L to 5 g/L stabilizer, 0.1 g/L to 5 g/L surfactant, 0.1 g/L to 5 g/L preservative; the buffer is selected from one or a combination of the following: tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer and trihydroxymethyl methane buffer; the buffer of the first reagent and the buffer of the second reagent are the same or different; the concentration of the buffer is 10 mmol/L to 500 mmol/L; the pH of the buffer is 6.5 to 8.0; the stabilizer is selected from the following one or a combination of: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000 and polyethylene glycol 8000; the surfactant is selected from the following one or a combination of: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide and AEO7; the preservative is selected from the following one or a combination of: azide, MIT, PC biological preservative and thimerosal; the azide is selected from the group consisting of: sodium azide, lithium azide and PC-300; the substrate comprises: glucose 6-phosphate and β-nicotinamide adenine dinucleotide.

10. A method of preparing a conjugate, comprising the steps of: 1) providing the glucose 6-phosphate dehydrogenase mutant of claim 1; 2) providing a hapten; 3) conjugating the glucose 6-phosphate dehydrogenase mutant with the hapten at a molar ratio of 1:3; step 1) and step 2) are in parallel or in interchangeable succession; the hapten has a molecular weight of 100 Da to 4000 Da.

11. The method of preparing a conjugate according to claim 10, comprising the steps of: 1) providing the glucose 6-phosphate dehydrogenase mutant; 2) providing the hapten or derivative thereof; 3) contacting the glucose 6-phosphate dehydrogenase mutant with the hapten or derivative thereof at 18° C. to 28° C. for 1 hour to 4 hours so that the hapten or derivative thereof is conjugated with the glucose 6-phosphate dehydrogenase mutant to obtain the conjugate; 4) optionally, purifying the conjugate; step 1) and step 2) are interchangeable or in parallel; the buffer is selected from the group consisting of: PBS, Tris, TAPS and TAPSO, The pH of the buffer is 6.0 to 8.0.

12. The glucose 6-phosphate dehydrogenase mutant of claim 1 which is shown in SEQ ID No.2.

13. The conjugate of claim 5, wherein the hapten is selected from the group consisting of: small molecule drugs, antibiotics, hormones, metabolites, polysaccharides, lipids and short peptides; the hapten has a molecular weight of 100 Da to 4000 Da.

14. The detection kit according to claim 9, comprising: a first reagent comprising: 100 mM to 300 mM buffer, 5 mM to 25 mM substrate, 0.01 μg/L to 1 mg/L anti-hapten antibody, 100 mM to 300 mM NaCl, 1 g/L to 5 g/L stabilizer, 1 g/L to 5 g/L surfactant, 1 g/L to 5 g/L preservative; a second reagent comprising: 100 mM to 300 mM buffer, 0.05 μg/L to 0.5 mg/L conjugate of claim 5, 1 g/L to 5 g/L stabilizer, 1 g/L to 5 g/L surfactant, 1 g/L to 5 g/L preservative; the stabilizer is bovine serum albumin; the surfactant is Tween20.

15. The method according to claim 10, wherein the hapten is selected from the group consisting of: small molecule drugs, antibiotics, hormones, metabolites, polysaccharides, lipids and short peptides.

16. The method according to claim 10, wherein the hapten has a molecular weight of 200 Da to 1500 Da.

17. The method of preparing a conjugate according to claim 10, comprising the steps of: 1) providing the glucose 6-phosphate dehydrogenase mutant in a buffer; 2) providing the hapten or derivative thereof in an aprotic solvent; 3) contacting the glucose 6-phosphate dehydrogenase mutant with the hapten or derivative thereof at 18° C. to 28° C. for 2 hours to 3 hours, so that the hapten or derivative thereof is conjugated with the glucose 6-phosphate dehydrogenase mutant to obtain the conjugate; 4) optionally, purifying the conjugate by desalting; step 1) and step 2) are interchangeable or in parallel; the buffer is selected from the group consisting of: PBS, Tris, TAPS and TAPSO, The pH of the buffer is 6.0 to 8.0; the aprotic solvent is selected from the following one or a combination of: acetonitrile, dimethylformamide and dimethyl sulfoxide; the glucose 6-phosphate dehydrogenase mutant comprises one or more free sulfhydryl groups prior to step 3); contacting the glucose 6-phosphate dehydrogenase mutant with the hapten or derivative thereof at a molar ratio of 1:n; wherein n is from 1 to 200.

18. The method of preparing a conjugate according to claim 17, wherein n is from 30 to 120.

Description

DESCRIPTION OF THE DRAWINGS

[0095] FIG. 1. G6PDH (wild-type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides of Leuconostoc spp.

[0096] FIG. 2. G6PDH mutant (SEQ ID No. 2).

DETAILED DESCRIPTION OF THE INVENTION

Examples

Example 1. Synthesis of Tobramycin Derivatives

[0097] Tobramycin (98 mg, 0.21 mmol) and Compound 1 (64 mg, 0.21 mmol) were dissolved in 5 mL of water, and stirred at room temperature for 5 h. The Tobramycin derivative were obtained by HPLC separation. The synthetic route was as follows:

##STR00003##

[0098] The structure of the synthesized product was confirmed by conventional methods. The effect of this embodiment is to make small molecule antigens (or haptens) bear a group that can bind to an enzyme, and the technical effect of the present application does not depend on the specific hapten derivative.

Example 2. Preparation of Mutants

[0099] The desired DNAs, for example, were synthesized by using well-known genetic engineering methods, inserted into an appropriate expression vector (such as E. coli expression vector), expressed in the expression host, and purified (such as affinity purification), resulting in the enzyme mutant shown in SEQ ID No. 2.

Example 3. Conjugation of Tobramycin Derivative to G6PDH Mutant

[0100] The G6PDH-Tobramycin conjugate of the present application was obtained in the following manner: the sulfhydryl-reactive group (such as maleimide group) on the tobramycin derivative molecule was covalently bound to the sulfhydryl group on the G6PDH molecule.

[0101] 4 μl of the solution containing the G6PDH enzyme mutant of Example 2 (or the control G6PDH enzyme mutant of the prior art) (5 mg/mL enzyme, 100 mmol PB, 100 mmol NaCl, pH=8.0), 200 μl PB solution, and 800 μl tobramycin derivative prepared in Example 1 were reacted with shaking at room temperature (18 to 28° C., preferably 20 to 25° C.) for 2.5 h.

[0102] After treated with desalting column (desalting solution: 100 mM PB, 0.1% NaN3, 1% NaCl, pH=8.0), the protein peaks were pooled to obtain the G6PDH-tobramycin conjugate.

Example 4. Preparation of the Kit

[0103] The kit for the detection of tobramycin was prepared, including:

[0104] 1. Preparation of the First Reagent: [0105] HEPES buffer 100 mM, pH 7.0 [0106] Anti-tobramycin antibody 0.5 μg/ml [0107] β-nicotinamide adenine dinucleotide, oxidized, 15 mmol/L [0108] Glucose 6-phosphate 15 mmol/L [0109] Bovine Serum Albumin 1 g/L [0110] Tween 20 1 g/L [0111] Sodium azide 1 g/L;

[0112] 2. Preparation of the Second Reagent: [0113] PB buffer 100 mM, pH8.0 [0114] G6PDH-tobramycin conjugate 0.1 μg/ml [0115] Bovine Serum Albumin 1 g/L [0116] Tween 20 1 g/L [0117] Sodium azide 1 g/L.

[0118] 3. Calibrator:

[0119] The pure tobramycin was diluted by buffer solution (100 mM HEPES buffer) to reach concentrations of 0, 0.6, 2.0, 4.0, 6.0, 10.0 mg/L (or added as needed);

[0120] 4. Quality Control:

[0121] The pure tobramycin was diluted by buffer solution (100 mM HEPES buffer) to reach concentrations of 1.5 mg/L, 3 mg/L, 8 mg/L (or added as needed).

Test Examples

[0122]

TABLE-US-00001 TABLE 1 Parameters of automatic biochemical analyzer Detector model Hitachi 7180 parameters Analysis point [Rate-A][10][20][24] WAVE(SUB/MAIN) [405] [340] S.VIL. [12.0] S.R1 [150] S.R3 [50] ABS.LIMIT: [32000] [increment] CALIB TYPE: [logit-log(3P)] POINT: [6] SPAN PONIT[6] Calibrator 0.0, 0.6, 2.0, 4.0, 6.0, 10.0 mg/L Samples Samples to be tested, such as serum, plasma, urine, saliva, cerebrospinal fluid, ascite, whole blood, secretion

Test Example 1. Calibration Absorbance of the Tobramycin Detection Kit

[0123]

TABLE-US-00002 TABLE 2 Calibration Absorbance of the Tobramycin Detection Kit Reagents of the present application Calibrator Read 1 Read 2 Mean S/S1 1 1832 1827 1829.5 151.93% 2 1935 1915 1925.0 105.22% 3 2132 2122 2127.0 110.49% 4 2377 2351 2364.0 111.14% 5 2519 2529 2524.0 106.77% 6 2779 2780 2779.5 110.12%

Test Example 2. Repeatability of the Tobramycin Detection Kit

[0124] High, medium and low quality control were repeatedly determined for 20 times. The repeatability CV of the kit of the present invention was less than 2.61%, indicating that the repeatability is favorable.

TABLE-US-00003 TABLE 3 Repeatability Test number Quality control 1 Quality control 2 Quality control 3 1 1.62 2.89 8.10 2 1.67 2.85 8.13 3 1.65 2.86 8.04 4 1.64 2.89 8.01 5 1.67 2.89 8.28 6 1.60 2.82 8.35 7 1.62 2.82 8.19 8 1.56 2.82 8.09 9 1.62 2.84 8.17 10 1.59 2.88 8.25 11 1.55 2.81 8.04 12 1.58 2.83 8.17 13 1.66 2.85 8.03 14 1.59 2.84 8.06 15 1.51 2.87 8.28 16 1.58 2.85 8.16 17 1.61 2.86 7.94 18 1.57 2.75 8.02 19 1.59 2.77 8.15 20 1.58 2.85 8.10 Mean 1.60 2.84 8.13 STD 0.04 0.04 0.11 CV 2.61% 1.31% 1.30%

Test Example 3. Linearity of the Tobramycin Detection Kit

[0125] The screened low-value and high-value samples were arithmetically diluted. Each sample was repeatedly tested for 3 times. The average value of the measured concentration and the theoretical concentration were analyzed to evaluate the recovery rate, indicating that the deviation of the results was less than 10%, and the linear performance reached 10 μg/ml.

TABLE-US-00004 TABLE 4 Linearity Measured Measured Measured Theoretical Relative Absolute value 1 value 2 value 3 Mean value deviation deviation 1 0.53 0.51 0.57 0.54 0.55 −0.02 −2.81% 2 1.51 1.48 1.53 1.51 1.53 −0.03 −1.73% 3 2.49 2.40 2.46 2.45 2.51 −0.06 −2.55% 4 3.46 3.48 3.43 3.46 3.50 −0.04 −1.10% 5 4.48 4.50 4.46 4.48 4.48 0.00 0.08% 6 5.53 5.33 5.47 5.44 5.46 −0.01 −0.25% 7 6.53 6.50 6.51 6.51 6.44 0.08 1.17% 8 7.61 7.60 7.61 7.61 7.42 0.19 2.53% 9 8.51 8.56 8.47 8.51 8.40 0.11 1.35% 10 9.56 9.48 9.48 9.51 9.38 0.13 1.34% 11 10.02 10.10 9.92 10.01 10.36 −0.35 −3.37%

Test Example 4. Accuracy

[0126] The pure tobramycin product of the US Pharmacopoeia was dissolved at various concentrations as stock solutions, and then equally diluted in the serum (diluted by at least 20 times) to prepare tobramycin solutions with different serum concentrations. The kit of the present invention was used to measure and calculate the deviation from the theoretical value. The results showed that the deviation of the recovery rate was less than 6%, and the accuracy was favorable.

TABLE-US-00005 TABLE 5 Accuracy Measured Measured Measured Absolute Relative USP value 1 value 2 value 3 Mean deviation deviation 1.00 1.06 1.08 1.04 1.06 0.06 6.00% 1.50 1.59 1.52 1.51 1.54 0.04 2.67% 2.00 2.02 1.98 2.04 2.01 0.01 0.67% 4.00 3.04 2.94 2.97 2.98 −0.02 −0.56% 8.00 5.09 4.95 4.99 5.01 0.01 0.20% 10.00 10.12 10.01 10.05 10.06 0.06 0.60%

Test Example 5. Antibody Inhibition Rate

[0127] 1. Detection Principle of Antibody Inhibition Rate

[0128] When the antibody binds to the G6PDH-tobramycin conjugate, the G6PDH enzyme activity is affected due to steric hindrance, which reduces the efficiency of the enzyme to catalyze the conversion of NAD to NADH. The difference between the experimental groups with and without the antibody can be compared by detecting the change in NADH amount, and such difference reflects the inhibitory ability of the antibody to G6PDH.

[0129] 2. Reaction System:

TABLE-US-00006 TABLE 6 Preparation of detection reagents for antibody inhibition rate RI (with antibody) Final concentration 0.1M PB/K (pH = 7.2) 0.1M G6P 15 mM P-NAD 15 mM Ab 1% R1 (without antibody) Final concentration 0.1M PB/K (pH = 7.2) 0.1M G6P 15 mM β-NAD 15 mM

TABLE-US-00007 TABLE 7 Detection parameters for antibody inhibition rate Detector model Hitachi 7180 analysis/time/point 2 point rate/10 min/10-15 points R1/S 150:25 Wavelength 405/340 (Sub/Primary) Type of reaction Increment

[0130] 3. Results:

[0131] The inhibition of G6PDH by the antibody could be obtained by comparing the absorbance of the G6PDH-tobramycin conjugate when the antibody was added or not.

[00001]$\mathrm{Antibody}\mathrm{inhibition}\mathrm{rate}=\left[1-\frac{\Delta A\left(\mathrm{with}\mathrm{antibody}\right)}{\Delta A\left(\mathrm{without}\mathrm{antibody}\right)}\right]*100\%$

[0132] where ΔA refers to the difference in absorbance between the two test time points on the reaction curve.

TABLE-US-00008 TABLE 8 Antibody inhibition rates of different G6PDH mutants (ΔA absorbance) 340 nm Group Group without with Inhibition G6PDH mutant antibody antibody rate K56C 2218 1983 10.60% D105C 1994 1925  3.46% D259C 1850 1674  9.51% D306C 2011 1992  0.94% D454C 2043 1891  7.44% D375C 2566 2523  1.68% G426C 2363 2302  2.58% A45C 2150 2133  0.79% K56C/D306C/D454C 1998 1145 42.69%

[0133] Although not limited to a specific theory, it can be partially explained that compared with the G6PDH mutants in the prior art, the mutation sites (i.e. the sites where free sulfhydryl groups are introduced) in the enzyme mutant of the present application (K56C/D306C/D454C) are the locations for conjugating with haptens (such as hormones, small molecule drugs, etc.). When the hapten binds to the hapten-specific antibody at these positions, the steric hindrance formed has the largest effect on the activity of the G6PDH enzyme, and at the same time, the steric folding of the molecule cannot be substantially affected after the mutations are introduced. Therefore, the location of these mutation sites is very important, and it is necessary to take into account the activity of the G6PDH enzyme, the spatial folding of the conjugate molecule, and also the adequate exposure of the hapten epitope.

[0134] Since the mutant of the enzyme has a significant increase in the antibody inhibition rate, it has obvious advantages in the calibration of the absorbance. After the conjugate formed by the enzyme mutant and the hapten is formulated into a kit, the performance of the reagent is significantly improved in terms of repeatability, total imprecision, linearity, and specificity.