CN110174363A - Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent - Google Patents

Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent Download PDF

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CN110174363A
CN110174363A CN201910423122.4A CN201910423122A CN110174363A CN 110174363 A CN110174363 A CN 110174363A CN 201910423122 A CN201910423122 A CN 201910423122A CN 110174363 A CN110174363 A CN 110174363A
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asp
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龚俊
祁金祥
肖兰萍
刘希
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Beijing Strong Biotechnologies Inc
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Beijing Strong Biotechnologies Inc
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Priority to CN201911365439.3A priority patent/CN111239060B/en
Priority to CN202310217235.5A priority patent/CN116359146A/en
Priority to CN202211151405.6A priority patent/CN115791649A/en
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Priority to CN202310452946.0A priority patent/CN116559425A/en
Priority to CN202010009771.2A priority patent/CN111504921B/en
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Priority to CN202310508418.2A priority patent/CN116381253A/en
Priority to CN202310554774.8A priority patent/CN116679047A/en
Priority to CN202310507880.0A priority patent/CN116754756A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

This application involves glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent.Specifically, the glucose-6-phosphate dehydrogenase mutant of the application includes one selected from the following mutation or combinations thereof: D306C, D375C, G426C compared to wild type glucose-6-phosphate dehydrogenase.Detection kit prepared by glucose-6-phosphate dehydrogenase mutant using the application, high specificity, high sensitivity, easy to operate, detection time is short, quantitative accurate, is suitble to high-throughput detection.

Description

Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent
Technical field
This application involves field of biological detection, more particularly to a kind of enzyme glucose-6-phosphate dehydrogenase of multisite mutation (abbreviation G6PDH) and its application in detection kit.
Background technique
Haptens, certain small-molecule substances (molecular weight is less than 4000Da), not can induce immune response individually, i.e., does not have Standby immunogenicity, but can get after the carriers such as itself and the poly-D-lysine of macro-molecular protein or nonantigenic are crosslinked or are combined Immunogenicity induces immune response.These small-molecule substances can have antigenicity, it only exempts from conjunction with response effect product Epidemic disease reactivity, does not have immunogenicity, also known as incomplete antigen.
Antigen-antibody reaction can occur in conjunction with corresponding antibody in haptens, and be unable to the generation of independent excitation human or animal body The antigen of antibody.It only has immunoreactivity, does not have immunogenicity, also known as incomplete antigen.Most of polysaccharide, lipoid, hormone, Small-molecule drug belongs to haptens.If chemically haptens and certain protein molecular (carrier) are combined, can obtain New immunogenicity, and animal can be stimulated to generate corresponding antibody.Haptens once and protein binding, just constitute the protein One epitope cluster.It is some smaller than general hapten molecule amount, but have specific structure chemical active radical substance (such as penicillin, Sulfa drug etc.), referred to as simple hapten.
Small molecule antigens or haptens cannot be pressed from both sides with double antibody due to a lack of can make more than two sites of sandwich method Heart method is measured, and mostly uses competitive mode.Principle is that the antigen and the competition of a certain amount of enzyme-labelled antigen in sample resist with solid phase Body combines.The more, the enzyme-labelled antigen being incorporated in solid phase is fewer for amount of antigen content in sample, develops the color more shallow.Small molecule hormone, medicine The ELISA such as object measurement is mostly used this method.
Glycocholic acid (Cholyglycin, CG) is cholic acid shape in conjunction with glycine as the specific example of haptens At mating type cholic acid, be one of main component of bile acid.Cholesterol passes through the enzymatic of a series of complex in liver cell Reaction forms primary bile acid, includes cholic acid (CA) and chenodeoxycholic acid (CDCA), in the steroids core of cholic acid there are three hydroxyl The hydroxyl of (C3, C7, C12), side chain terminal are combined into glycocholic acid (Fig. 1) with peptide bond and glycine.
Glycocholic acid is synthesized by liver cell, is discharged into gall-bladder through bile capillaries, bile duct, is entered duodenum in company with bile, is helped Fatty digestion and absorption in food.95% bile acid is in ileum and colon by reabsorption, and trans-portal vein returns liver again, by liver cell Intake recycles, and re-absorbed glycocholic acid enters liver-intestines circulation again, and by this mechanism, body can make full use of glycocholic acid.
Under normal circumstances, the content of cholic acid and its little in peripheral blood, normal person either on an empty stomach or postprandial, Content of glycocholic acid in peripheral blood is in extremely low level.It, will when human hepatocyte is impaired or cholestasis Cause glycocholic acid metabolism and disturbance of circulation, makes the ability decline of liver cell intake glycocholic acid, lead to content of glycocholic acid liter in blood Height, and glycocholic acid value height is related to the severity of hepatocellular damage and bile acid biosynthesis obstacle.
The content of glycocholic acid is to evaluate the sensitive indicator of hepatocyte function and liver and gallbladder system substance circulatory function in measurement serum One of.With ALT, AST, total bilirubin (TBIL), alkaline phosphatase (ALP), paddy acyl transpeptidase (GGT), seralbumin (ALB) It is compared Deng routine Liver function grade, the measurement of glycocholic acid is more sensitive.Therefore, in chronic hepatitis, oxyhepatitis, cirrhosis, liver In the detection of the liver functions such as cancer, obstructive liver disease, hepato-enteric circulation obstacle, bile duct, gall-bladder excretory function obstacle, glycocholic acid be can be used as more Good Testing index.
The glycocholic acid detection method being currently known mainly has: radio immunoassay, enzyme-linked immunization, chemiluminescence immunoassay Analytic approach, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry etc..But these detection methods exist More defect, such as radio immunoassay isotope has that radioactive pollution, validity period be shorter, many disadvantages inconvenient for operation End, enzyme linked immunosorbent assay operation is relatively complicated, takes a long time, and is not suitable for clinically using.Chemiluminescence is although sensitivity Preferably, but matched special equipment is needed, the higher cost that comes into operation is unfavorable for promoting.During clinical detection diagnosis, with Based on homogeneous enzyme immunoassay method (EMIT) and the detection of latex enhancing immune turbidimetry.
The principle of homogeneous EIA: in liquid homogeneous phase reaction system, enzyme-labelled antigen (such as G6PDH-CG) with it is non- Labelled antigen (CG), competition is combined with quantitative antibody (CG antibody), when antibody is more in conjunction with non-labeled antigen, enzyme mark Remember that the activity of antigen release is more, substrate for enzymatic activity NAD+ generation NADH is more, and the suction of NADH is detected under 340nm wavelength Light varience can extrapolate the content of CG in liquid.
Summary of the invention
This application claims 201910017764.4 priority (priority date on January 9th, 2019).
In view of the demand of this field, this application provides a kind of novel glucose-6-phosphate dehydrogenase mutant and its Preparing the purposes in glycocholic acid detection kit.
According to some embodiments, a kind of glucose-6-phosphate dehydrogenase mutant is provided.It is different to have and deliver 20,000,507 6033890 (Homogeneous immunoassays using of patent United States Patent Mutant glucose-6-phosphate dehydrogenases Edward Benjamin Jakobovits etc.) 6 phosphorus The mutant of sour grapes glucocorticoid dehydrogenase, the glucose-6-phosphate dehydrogenase mutant of the application, it includes mutation selected from the following: D306C、G426C、D375C。
According to some embodiments, a kind of glucose-6-phosphate dehydrogenase mutant, the glucose 6-phosphate are provided Dehydrogenase mutant is shown in sequence selected from the following: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.
According to some embodiments, a kind of polynucleotides are provided, encode the glucose-6-phosphate dehydrogenase of the application Mutant.
According to some embodiments, a kind of expression vector is provided, it includes the polynucleotides of the application.
According to some embodiments, a kind of host cell is provided, it includes the expression vectors of the application.Host cell can To be protokaryon (such as bacterium) or eukaryon (such as yeast).
According to some embodiments, a kind of conjugate is provided, is the glucose-6-phosphate dehydrogenase mutation of the application Body and haptens are coupled according to molar ratio 1:n.
In some embodiments, n is 1 to 50, for example, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15, 16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、 41、42、43、44、45、46、47、48、49、50。
In some specific embodiments, the glucose-6-phosphate dehydrogenase mutant and haptens of the application are according to rubbing You are than being preferably 1:30.
250 in some specific embodiments, the molecular weight of haptens is 200Da to 4000Da, such as: 200, 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 application, the skilled person will understand that, " haptens " also includes the form of its derivative.For the ease of with 6- phosphorus Sour grapes glucocorticoid dehydrogenase is coupled, for those itself without the half of coupling group (for example, the group reacted with sulfydryl) Antigen (such as CG), can it is engineered and have connector, so as to sulfydryl covalent bond.Therefore, in this application, haptens spreads out Biology refers to, haptens that is engineered and having sulfhydryl reactive group.
Haptens is selected from: small-molecule drug (such as antibiotic, psychotropic agent), hormone, metabolin, sugar, lipid, amino acid.
Haptens is such as, but not limited to: vitamin D, 25 hydroxyvitamin D, 1,25 pairs of hydroxyvitamin D, folic acid, cardiac glycoside, enzymes Phenolic acid, Lei Paming, cyclosporin A, ethamine iodine answer ketone, methotrexate, tacrolimus, serum amino acid, bile acid, glycocholic acid, Phenylalanine, ethyl alcohol, urine Ni Keding metabolite Ke are for peaceful, urine morphine, the single hydroxyl amphyl of urine, aging changes, blood plasma Sweet C prime, histamine, thyrotropic hormone, prolactin(PRL, galactagogin, growth hormone, flitropin, promotees Huang at polyamines Body generates element, corticotropin, antidiuretic hormone, calcitonin, Procalcitonin, parathyroid hormone, thyroxine, three Iodine first shape original propylhomoserin, anti-triiodo first shape original propylhomoserin, free thyroxine, free triiodo first shape original propylhomoserin, cortisol, urine 17- hydroxyl Corticosteroid, urine 17-ketosteroid, dehydrobenzene and sulfuric ester, aldosterone, urine vanilla mandelic acid, plasma renin, blood Angiotensin, hematopoietin, testosterone, protona, androstenedione, 17 α hydroxyprogesterones, oestrone, estriol, estradiol, Progesterone, human chorionic gonadotrophin, insulin, proinsulin, C peptide, gastrin, Plasma Prostaglandin, blood plasma 6- ketone forefront Parathyrine F1 α, prostacyclin, adrenaline, catecholamine, norepinephrine, element, cyclic adenosine monophosphate, ring are received cholecystokinin, Guanosine 5-monophosphate, vasoactive peptide, growth hormone release inhibiting hormone, secretin, P- substance, neurotensin, thromboxane A2, thromboxane B2,5 hydroxyls Tryptamines, neuropeptide tyrosine, osteocalcin.
In specific embodiments, haptens is glycocholic acid or derivatives thereof.Although specific using glycocholic acid as one Example, but technical staff is appreciated that specific type of the technical effect independent of haptens of the application, it is suitable for Any haptens that immunology detection can be carried out by competition law.
In specific embodiments, haptens is glycocholic acid derivative, has sulfhydryl reactive group, such as comes acyl Asia Amine, acetyl bromide, vinyl sulfone or aziridine.In specific embodiments, haptens is glycocholic acid derivative, such as Formulas I institute Show:
According to some embodiments, a kind of reagent is provided, it includes the conjugates of the application.
According to some embodiments, the glucose-6-phosphate dehydrogenase mutant of the application is provided in preparation detection reagent In purposes.
According to some embodiments, purposes of the conjugate of the application in preparation detection reagent is provided.
In specific embodiments, the detection reagent is selected from: enzyme-linked immunization detection reagent, chemiluminescence immunoassay Method detection reagent, homogeneous enzyme immunoassay method detection reagent, latex enhancing immune turbidimetry detection reagent.
In specific embodiments, the detection reagent is preferably based on the reagent of competition law detection.
According to some embodiments, a kind of glycocholic acid detection kit is provided, it includes:
- the first reagent, first reagent include substrate and glycocholic acid antibody；The substrate is glucose 6-phosphate dehydrogenation The substrate of enzyme；
- the second reagent, second reagent include the conjugate of the application；
Optionally, calibration object, the calibration object include 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid；With And
Optionally, quality-control product, the quality-control product include 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid.
According to one embodiment, a kind of glycocholic acid detection kit is provided, it includes:
First reagent, it includes:
10mM to 500mM buffer,
0.5g/L to 5g/L substrate,
The glycocholic acid antibody of 0.1mg/L to 1mg/L,
10mM to 300mM NaCl,
0.1g/L to 5g/L stabilizer,
0.1g/L to 5g/L surfactant,
0.1g/L is to 5g/L preservative；
Second reagent, it includes:
10mM to 500mM buffer,
Conjugate described in claim 5,
0.1g/L to 5g/L stabilizer,
0.1g/L to 5g/L surfactant,
0.1g/L is to 5g/L preservative.
In some embodiments, the buffer is selected from the following a kind of or combines: tromethamine buffer, phosphoric acid Salt buffer, Tris-HCl buffer, citric acid-sodium citrate buffer solution, barbitol buffer solution, glycine buffer, boric acid Salt buffer, Pehanorm buffer；It is preferred that phosphate buffer；The concentration of the buffer be 10mmol/L extremely 500mmol/L, preferably 100mM；The pH of the buffer is 6.5 to 7.5, preferably 7.2 or 7.0.
In some embodiments, the stabilizer is selected from the following a kind of or combination: bovine serum albumin(BSA), trehalose, Glycerol, sucrose, mannitol, glycine, arginine, Macrogol 6000, PEG 8000；It is preferred that bovine serum albumin(BSA).
In some embodiments, the surfactant is selected from the following a kind of or combines: Brij35, Tritiom X- 100, Tritiom X-405, Tween20, Tween30, Tween80, cocoanut fatty acid diethanolamide, AEO7, preferably Tween20。
In some embodiments, the preservative is selected from the following a kind of or combines: azide, MIT, PC- 300, thimerosal；The azide is selected from: Sodium azide, nitrine lithium.
In some embodiments, the substrate includes: 6- phosphoglucose sugar, β-nicotinamide adenine dinucleotide.
In some embodiments, the concentration of buffer is 100mM.
In some embodiments, the concentration of substrate of G6PDH enzymic catalytic reaction is 5g/L.
In some embodiments, the concentration of glycocholic acid antibody is 0.1mg/L.
In some embodiments, the concentration of NaCl is 300mM.
In some embodiments, the concentration of stabilizer is 0.5g/L.
In some embodiments, the concentration of surfactant is 0.1g/L.
In some embodiments, the concentration of preservative is 1g/L.
Detailed description of the invention
Fig. 1 glycocholic acid structure chart.
Fig. 2 glycocholic acid derivant structure figure.
Fig. 3 A.G6PDH (wild type) amino acid sequence (SEQ ID No.1)；From the bright beading of Leuconostoc vacation goldbeater's skin Bacterium Leuconostoc pseudomesenteroides.
Fig. 3 B.G6PDH (D306C) amino acid sequence (SEQ ID No.2).
Fig. 3 C.G6PDH (D375C) amino acid sequence (SEQ ID No.3).
Fig. 3 D.G6PDH (G426C) amino acid sequence (SEQ ID No.4).
Specific embodiment
Embodiment
The synthesis of 1. glycocholic acid derivative of embodiment
Into the 25mL two-mouth bottle of dried and clean be added glycocholic acid (1.0eq), dimaleoyl imino ethamine (1.0g, 1.0eq), triethylamine (3.0eq)；
It adds dimethylformamide (5mL) to stir to Quan Rong, be added dichloroethanes (1.25eq), in 25 DEG C of stirring 2h；
HPLC monitoring, until end of reaction；
Above-mentioned reaction mixture is added in water (25mL), ethyl acetate 20mL × 3 are added and are extracted；
Merge organic phase, anhydrous Na2SO4It is dry, it is concentrated under reduced pressure, gained grease is purified to obtain 1.04g cream with column chromatography White powdery solids, yield 45%, M+:602.72.
The effect of the present embodiment is so that the group that CG can be combined with one with enzyme, the technical effect of the application is not Dependent on specific hapten derivant.
The coupling of embodiment 2. glycocholic acid derivative and G6PDH molecule
According to the G6PDH- glycocholic acid conjugate of the application, it is coupled in the following way: glycocholic acid derivative molecular On sulfydryl reactive group (such as maleimide base group) and G6PDH molecule on sulfydryl covalent bond.
1. solution is prepared:
Glycocholic acid derivative solution: glycocholic acid derivative 10mg/ml prepared by embodiment 1 is dissolved in DMF；
G6PDH solution: 6.7mg/mL G6PDH (mutant or wild type of the application), PB 100mmol, NaCl 100mmol, pH=8.0；
Conjugate solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH=7.2；
Desalting soln: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH=7.2.
2. COUPLING PROCEDURE: 1.6ml G6PDH solution, 6ml conjugate solution and 0.40ml glycocholic acid derivative solution, in room temperature (20 to 25 DEG C) reaction 4h.
3. being eluted with above-mentioned desalting soln using desalting column after above-mentioned reaction system shaken at room temperature is reacted 4h, receive Collect protein peak, products therefrom, that is, G6PDH- glycocholic acid conjugate.
The preparation of 3. kit of embodiment
The kit of the following detection glycocholic acid of preparation, it includes:
Reagent R1 includes:
100mM PB buffer, pH 7.2
15mM glucose 6-phosphate
15mM β-nicotinamide adenine dinucleotide
0.1mg/L glycocholic acid antibody
200mM NaCl
0.5g/L bovine serum albumin(BSA)
0.1g/L Tween20
1g/L Sodium azide；
Reagent R2, comprising:
100mM PB buffer, pH 7.2
0.1mg/L G6PDH-CG conjugate
0.5g/L bovine serum albumin(BSA)
0.1g/L Tween 20
1g/L Sodium azide；
Calibration object: 100mM PB buffer, pH 7.2 and 0,2.5,5.0,10,20,40mg/L glycocholic acid (or on demand It is added)；
Quality-control product: 100mM PB buffer, pH 7.2 and 1.5,8.0,25,35mg/L glycocholic acid (or being added on demand).
Detect example
Reaction time: 10min, wherein incubation time is 4.7min, and after being incubated for 1min after addition reagent R2, measurement, which is read, inhales Luminosity A1, after being incubated for 1min, absorbance A 2 is read in measurement, calculates Δ A=(A2-A1)/min.Sample is calculated by calibration curve The content of glycocholic acid in this: CG=sample cell absorbance * calibration object concentration/calibration object absorbance.
Performance detection is carried out to the glycocholic acid detection kit prepared in embodiment 3, main detection performance is total not smart Density, repeatability, recycling, linear, specificity etc..
1. full automatic biochemical apparatus parameter of table
Detect type Hitachi 7180
Analysis/time/point 2 point velocities/10min/20-24 point
R1/R2/S 120:40:9
Wavelength (secondary/main) 405/340
Reaction type It is incremented by
Type of calibration Spine type
Calibration point 6
Calibration object concentration 0/2.50/5.00/10.00/20.00/40.00
1. glycocholic acid detection kit of test case calibrates absorbance
2. glycocholic acid detection kit of table calibrates absorbance
Detect the 2. total imprecision of glycocholic acid detection kit of example
The total imprecision of table 3.
Detect 3. glycocholic acid detection kit repeatability of example
4. repeatability of table
Detect the recycling of 4. glycocholic acid detection kit of example
Table 5. recycles
It is linear to detect 5. glycocholic acid detection kit of example
Table 6. is linear
Detect the 6. anti-specificity of glycocholic acid detection kit of example
7. specificity of table
Chaff interferent (40 μ g/ml) The application reagent (D306C) Contrast agents (A45C mutant)
Glycodesoxycholic acid 18.61% 30.20%
Glycochenodeoxycholate 1.63% 37.91%
Chenodeoxycholic acid 0.61% 16.28%
Ursodesoxycholic acid - 0.42% 6.55%
Sodium taurocholate 61.21% 61.90%
NaTDC 4.22% 11.74%
The analogue cross reaction very little of the application reagent and glycocholic acid even without.
Detect 7. antibody inhibiting rate of example
1. the testing principle of antibody inhibiting rate
When antibody is in conjunction with G6PDH-CG conjugate, since steric hindrance causes G6PDH enzymatic activity to be affected, thus So that the efficiency that its catalysis NAD is converted into NADH reduces, by detecting the variation of NADH amount, antibody is added and does not add to compare Enter the difference of the experimental group of antibody, this species diversity is presented as antibody to the rejection ability of G6PDH.
2. reaction system:
It is prepared by the detection reagent of 8. antibody inhibiting rate of table
9. antibody inhibiting rate of table detects set factors
Detect type Hitachi 7180
Analysis/time/point 2 point velocities/10min/10-15 point
R1/S 120:20
Wavelength (secondary/main) 405/340
Reaction type It is incremented by
3. result:
When comparing addition antibody and antibody not being added, G6PDH-CG conjugate absorbance measured value is detected respectively, can be obtained Inhibition situation of the antibody to G6PDH.
The absorbance of G6PDH-CG when the antibody inhibiting rate absorbance change value of G6PDH-CG=when containing antibody/is free of antibody Changing value.
Relative to the mutational site delivered, the mutant of the application is significantly improved on antibody inhibiting rate, can reach 30% or more, up to 50%.And the inhibiting rate of common mutational site (such as A45C, K55C) only has 20% or so very before To lower.
The antibody inhibiting rate of 10. difference G6PDH mutant of table
Although being not limited to specific theory, can partly explain are as follows: and G6PDH mutant phase in the prior art Than mutational site (introducing the site of free sulfhydryl groups) is resisted with half in the application enzyme mutant (D306C, D375C, G426C) Where the position that former (such as hormone, small-molecule drug etc.) is coupled.Haptens on this position with hapten specificity When antibody combines, the steric hindrance constituted is minimum to the activity influence of G6PDH enzyme, while after introducing mutation, can't be real The space folding of molecule is influenced in matter.Therefore, the position in this mutational site is extremely important, needs to combine G6PDH enzyme The abundant exposure of activity, the space folding of coupling molecule and haptens epitope.
Since the mutant of enzyme is significantly improved on antibody inhibiting rate, can have in calibration absorbance apparent excellent Gesture.After conjugate after the mutant and hapten conjugation of enzyme is configured to kit, since the improvement of calibration curve becomes, reagent There is apparent performance boost in aspect of performance such as repeatability, total imprecision, linear, specificity.
Sequence table
<110>Beijing Strong Biotechnologies, Inc.
<120>glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent
<130> 390017CG-390010
<150> 201910017764.4
<151> 2019-01-09
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 486
<212> PRT
<213>Leuconostoc leuconostoc pseudomesenteroides (Leuconostoc pseudomesenteroides)
<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, compared to wild type, 306 D replace with C
<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
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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, compared to wild type, 375 D replace with C
<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, compared to wild type, 426 G replace with C
<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
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35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
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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
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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 (10)

1. a kind of glucose-6-phosphate dehydrogenase mutant, compared to wild type glucose-6-phosphate dehydrogenase, it includes be selected from Mutation below or combinations thereof: D306C, D375C, G426C；
Preferably, the glucose-6-phosphate dehydrogenase mutant is shown in sequence selected from the following: SEQ ID No.2, SEQ ID No.3、SEQ ID No.4。
2. a kind of polynucleotides encode glucose-6-phosphate dehydrogenase mutant described in claim 1.
3. a kind of expression vector, it includes polynucleotides as claimed in claim 2.
4. a kind of host cell, it includes expression vectors as claimed in claim 3.
5. a kind of conjugate, be glucose-6-phosphate dehydrogenase mutant and haptens described in claim 1 according to mole It is coupled than 1:n；
N is 1 to 50, preferably 10,15,20,25,30,35,40,45 or 50；
More preferable n is 30；
It is preferred that the haptens is selected from: antibiotic, hormone, metabolin, sugar, lipid, amino acid；
The molecular weight of the haptens is 200Da to 4000Da, preferably 200Da to 1500Da；
It is further preferred that the haptens is glycocholic acid or derivatives thereof.
6. a kind of reagent, it includes the conjugates described in claim 5.
7. the purposes any one of selected from the following in preparation detection reagent:
Conjugate described in glucose-6-phosphate dehydrogenase mutant described in claim 1, claim 5；
Preferably, the detection reagent is the detection reagent of haptens；
Preferably, the detection reagent is selected from: enzyme-linked immunization detection reagent, Chemiluminescence immunoassay detection reagent, homogeneous enzyme Immunization detection reagent, latex enhancing immune turbidimetry detection reagent；
Preferably, the haptens is selected from: antibiotic, hormone, metabolin, sugar, lipid, amino acid；
The molecular weight of the haptens is 200Da to 4000Da, preferably 200Da to 1500Da.
8. a kind of glycocholic acid detection kit, it includes:
First reagent, first reagent include substrate, glycocholic acid antibody；
Second reagent, second reagent include conjugate described in claim 5；
Optionally, calibration object, the calibration object include 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid；And
Optionally, quality-control product, the quality-control product include 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid.
9. glycocholic acid detection kit according to claim 8, it includes:
First reagent, it includes:
10mM to 500mM buffer,
0.5g/L to 5g/L substrate,
The glycocholic acid antibody of 0.1mg/L to 1mg/L,
10mM to 300mM NaCl,
0.1g/L to 5g/L stabilizer,
0.1g/L to 5g/L surfactant,
0.1g/L is to 5g/L preservative；
Second reagent, it includes:
10mM to 500mM buffer,
Conjugate described in claim 5,
0.1g/L to 5g/L stabilizer,
0.1g/L to 5g/L surfactant,
0.1g/L is to 5g/L preservative；
The buffer is selected from the following a kind of or combines: tromethamine buffer, phosphate buffer, Tris-HCl buffering Liquid, citric acid-sodium citrate buffer solution, barbitol buffer solution, glycine buffer, borate buffer solution, Pehanorm Buffer；It is preferred that phosphate buffer；
The concentration of the buffer is 10mmol/L to 500mmol/L；
The pH of the buffer is 6.5 to 7.5；
The stabilizer is selected from the following a kind of or combines: bovine serum albumin(BSA), trehalose, glycerol, sucrose, mannitol, sweet ammonia Acid, arginine, Macrogol 6000, PEG 8000；It is preferred that bovine serum albumin(BSA)；
The surfactant is selected from the following a kind of or combination: Brij35, Tritiom X-100, Tritiom X-405, Tween20, Tween30, Tween80, cocoanut fatty acid diethanolamide, AEO7, preferably Tween20；
The preservative is selected from the following a kind of or combines: azide, MIT, PC-300, thimerosal；
The azide is selected from: Sodium azide, nitrine lithium；
The substrate includes: 6- phosphoglucose sugar, β-nicotinamide adenine dinucleotide.
10. a kind of preparation method of conjugate comprising step:
Glucose-6-phosphate dehydrogenase mutant described in claim 1 is provided；
Haptens is provided, the haptens is selected from: antibiotic, hormone, metabolin, sugar, lipid, amino acid；
The molecular weight of the haptens is 200Da to 4000Da, preferably 200Da to 1500Da；
The glucose-6-phosphate dehydrogenase mutant and the haptens are coupled according to molar ratio 1:n；
N is 1 to 50, preferably 10,15,20,25,30,35,40,45 or 50；
It is further preferred that n is 30；
It is further preferred that the haptens is glycocholic acid or derivatives thereof.
CN201910423122.4A 2019-01-09 2019-05-21 Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent Pending CN110174363A (en)

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CN202310257027.8A CN116144619A (en) 2019-01-09 2019-12-26 Theophylline detection kit
CN201911365439.3A CN111239060B (en) 2019-01-09 2019-12-26 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent
CN202310217235.5A CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
CN202211151405.6A CN115791649A (en) 2019-01-09 2019-12-27 Glycocholic acid detection kit
CN201911372147.2A CN112285037B (en) 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
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CN202310811212.7A CN116735512A (en) 2019-01-09 2019-12-27 Use of conjugates in the preparation of detection reagents
CN202211153004.4A CN115808398A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202310811210.8A CN116626281A (en) 2019-01-09 2019-12-27 Digitoxin detection kit
CN202211151264.8A CN116008201A (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent
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CN202310726493.6A CN116773795A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
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CN202010009771.2A CN111504921B (en) 2019-01-09 2020-01-06 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of gentamicin detection reagent
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CN201910423122.4A Pending CN110174363A (en) 2019-01-09 2019-05-21 Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent
CN201911365439.3A Active CN111239060B (en) 2019-01-09 2019-12-26 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent
CN202310257027.8A Pending CN116144619A (en) 2019-01-09 2019-12-26 Theophylline detection kit
CN202310217235.5A Pending CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
CN202211153004.4A Pending CN115808398A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202310811210.8A Pending CN116626281A (en) 2019-01-09 2019-12-27 Digitoxin detection kit
CN202211151405.6A Pending CN115791649A (en) 2019-01-09 2019-12-27 Glycocholic acid detection kit
CN202310811212.7A Pending CN116735512A (en) 2019-01-09 2019-12-27 Use of conjugates in the preparation of detection reagents
CN202211151264.8A Pending CN116008201A (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent
CN201911372535.0A Active CN112285038B (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of digitoxin detection reagent
CN201911372147.2A Active CN112285037B (en) 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
CN202310811498.9A Pending CN116698772A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
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CN202310724066.4A Pending CN116773827A (en) 2019-01-09 2019-12-31 Use of conjugates in the preparation of detection reagents
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CN201911365439.3A Active CN111239060B (en) 2019-01-09 2019-12-26 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent
CN202310257027.8A Pending CN116144619A (en) 2019-01-09 2019-12-26 Theophylline detection kit
CN202310217235.5A Pending CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
CN202211153004.4A Pending CN115808398A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202310811210.8A Pending CN116626281A (en) 2019-01-09 2019-12-27 Digitoxin detection kit
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CN202310811212.7A Pending CN116735512A (en) 2019-01-09 2019-12-27 Use of conjugates in the preparation of detection reagents
CN202211151264.8A Pending CN116008201A (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent
CN201911372535.0A Active CN112285038B (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of digitoxin detection reagent
CN201911372147.2A Active CN112285037B (en) 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
CN202310811498.9A Pending CN116698772A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
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CN202310364997.8A Pending CN116298257A (en) 2019-01-09 2019-12-31 Phenytoin detection kit
CN202310365160.5A Pending CN116355873A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
CN202310726493.6A Pending CN116773795A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
CN202310364266.3A Pending CN116718764A (en) 2019-01-09 2019-12-31 Use of conjugate in preparation of phenytoin detection reagent
CN201911404154.6A Active CN111504920B (en) 2019-01-09 2019-12-31 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent
CN202310724066.4A Pending CN116773827A (en) 2019-01-09 2019-12-31 Use of conjugates in the preparation of detection reagents
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CN202310810479.4A Pending CN116819060A (en) 2019-01-09 2020-01-03 Digoxin detection kit
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CN202310318754.0A Pending CN116124721A (en) 2019-01-09 2020-01-06 Gentamicin detection kit
CN202310452740.8A Pending CN116430056A (en) 2019-01-09 2020-01-06 Method for preparing conjugate
CN202310453290.4A Pending CN116338215A (en) 2019-01-09 2020-01-06 Tacrolimus detection kit
CN202310320729.6A Pending CN116297271A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of kits
CN202310320137.4A Pending CN116148198A (en) 2019-01-09 2020-01-06 Preparation method of gentamicin detection reagent
CN202310452946.0A Pending CN116559425A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of detection reagents
CN202310554774.8A Pending CN116679047A (en) 2019-01-09 2020-01-07 Method for preparing conjugate
CN202310553479.0A Pending CN116699125A (en) 2019-01-09 2020-01-07 Use of conjugates in the preparation of detection reagents
CN202310555230.3A Pending CN116718761A (en) 2019-01-09 2020-01-07 Cyclosporine A detection kit
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CN202010013644.XA Active CN111487208B (en) 2019-01-09 2020-01-07 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of methotrexate detection reagent
CN202310507880.0A Pending CN116754756A (en) 2019-01-09 2020-01-07 Methotrexate detection kit
CN202310508217.2A Pending CN116298330A (en) 2019-01-09 2020-01-07 Use of conjugates in the preparation of detection reagents
CN202310508418.2A Pending CN116381253A (en) 2019-01-09 2020-01-07 Method for preparing conjugate
CN202310725902.0A Pending CN116577494A (en) 2019-01-09 2020-01-08 Use of conjugates in the preparation of detection reagents
CN202310702858.1A Pending CN116859035A (en) 2019-01-09 2020-01-08 Use of conjugates in the preparation of detection reagents
CN202010017376.9A Active CN111537452B (en) 2019-01-09 2020-01-08 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of amikacin detection reagent
CN202310726230.5A Pending CN116577495A (en) 2019-01-09 2020-01-08 Method for preparing conjugate
CN202010016535.3A Active CN111693473B (en) 2019-01-09 2020-01-08 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of rapamycin detection reagent
CN202310726069.1A Pending CN116754761A (en) 2019-01-09 2020-01-08 Amikacin detection kit
CN202310702860.9A Pending CN116699122A (en) 2019-01-09 2020-01-08 Rapamycin detection kit
CN202310702870.2A Pending CN116840462A (en) 2019-01-09 2020-01-08 Method for preparing conjugate

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CN111487206A (en) * 2019-01-09 2020-08-04 北京九强生物技术股份有限公司 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent
CN111487208A (en) * 2019-01-09 2020-08-04 北京九强生物技术股份有限公司 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of methotrexate detection reagent
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CN113046335A (en) * 2019-12-27 2021-06-29 中国科学院天津工业生物技术研究所 Glucose 6-phosphate dehydrogenase mutant with bionic coenzyme preference and application thereof
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