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Fructose amino acid oxidase, preparation method and glycatedalbumin detection kit comprising oxidase

A fructose amino acid and glycosylated albumin technology, applied in biochemical equipment and methods, redox enzymes, botanical equipment and methods, etc., can solve the problems of unsatisfactory stability, poor thermal stability, unfavorable application of biochemical instruments, etc.

Active Publication Date: 2014-04-02
NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is precisely because of the poor thermal stability of the existing fructose amino acid oxidase that the stability of the glycated albumin detection kits currently on the market cannot meet the requirements. In many cases, a large amount of stabilizers are added to the kits , to improve the stability of the kit (International Patent Application Publication No. WO2002061119A1), which leads to high viscosity of the reagent and a series of other problems, which are not conducive to the application in biochemical instruments

Method used

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  • Fructose amino acid oxidase, preparation method and glycatedalbumin detection kit comprising oxidase
  • Fructose amino acid oxidase, preparation method and glycatedalbumin detection kit comprising oxidase
  • Fructose amino acid oxidase, preparation method and glycatedalbumin detection kit comprising oxidase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Mutation library construction

[0079]1. The sequence shown in Sequence 2 is synthesized by the method of total gene synthesis, and cloned into the pET-22b vector, and the restriction sites used are NdeI and XhoI. The thus obtained plasmid pET-Ama was used as a template for the following error-prone PCR and WHOP-PCR.

[0080] 2. Error-prone PCR reaction system and conditions

[0081] The reaction system in Table 1 is 100ul:

[0082] name

Volume (ul)

dNTP Mixture (2.5mM each)

8

dTTP (100mM)

0.8

dCTP (100mM)

0.8

10*PCRBuffer

10

Upstream primer (5mM), Sequence 3

20

Downstream primer (5mM), sequence 4

20

[0083] MnCl 2 (5mM)

10

Mg 2+ (25mM)

14

Taq enzyme (5U / ul)

1

Template (10ng / ul)

5

water

12

[0084] The reaction conditions are:

[0085] 95°C for 5min; 94°C for 30sec; 55°C for 30sec; 72°C for 2min; 30 cycles; 72°C ...

Embodiment 2

[0097] Screening of mutant libraries

[0098] 1. Culture, induction and expression

[0099] After mixing the obtained clones with a coating stick, they were collected in a centrifuge tube, and the plasmid was extracted, and the obtained plasmid was transformed into BL21 (ED3) for the next step of screening. Next, the above-mentioned transformant was inoculated in a 96-well plate, wherein the last well was inoculated with a wild-type strain as a control, and the medium used was 150ulLB / well containing ampicillin antibiotic, and the orifice plate was used as a retention plate. The next day, transfer to another 96-well plate in the same order. The medium used is 150ulLB / well, and ampicillin antibiotics and IPTG are added for induction at the same time. Cultivate at 37°C for 6 hours, collect bacteria by centrifugation at 3800rpm, and remove the medium. This orifice plate serves as the assay plate.

[0100] 2. Screening

[0101] Add 150ul of lysis solution (100mM Tris, pH8.0; 0....

Embodiment 3

[0104] Enzyme Activity Determination and Thermal Stability Analysis of Fructose Amino Acid Oxidase

[0105] The purified fructose amino acid oxidase was diluted to about 10 ug / ml in 100 mM Tris, pH 8.0 buffer.

[0106] Take 50ul of the fructose amino acid oxidase in a 96-well PCR, heat-treat at 50°C for 15min, and store at 4°C.

[0107] Transfer 50ul of heat-treated fructose amino acid oxidase to a 96-well plate, and take 50ul of unheated fructose amino acid oxidase on the same 96-well plate. Incubate at 37°C for 10 minutes.

[0108] Add the chromogenic solution (Tris, 100mM, pH8.0; TOOS solution, 15mM; 4-APP, 0.5mM; POD, 40U / ml; fructoselysine, 15mM) that has been incubated to 37°C beforehand, and incubate at 37°C React for 30 minutes.

[0109] The absorbance at 555 nm was recorded using a microplate reader.

[0110] Divide the absorbance value of the heat-treated fructose amino acid oxidase by the absorbance value of the non-heat-treated fructose amino acid oxidase, and ...

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Abstract

The invention discloses fructose amino acid oxidase which has an amino acid sequence shown as SEQID.No.1 (sequence identifier number 1) or has above 80% homology with the amino acid sequence. One or more amino acid residues in corresponding positions of amino acid selected from (a) to (f) are substituted. The obtained fructose amino acid oxidase has higher thermostability: (a) 59-site glutamic acid, (b) 98-site glutamic acid, (c) 225-site glycine, (d) 277-site lysine, (e) 283-site glutamic acid and (f) 355-site aspartic acid. The invention further discloses a preparation method of the oxidase and a kit comprising the oxidase and used for determining glycatedalbumin. The kit has higher thermostability and can accurately determine the glycatedalbumin.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis, and specifically provides a fructose lysine oxidase mutant with high thermal stability, a method for measuring glycated albumin, and a kit for measuring glycated albumin. The invention can be used in clinical testing and can accurately measure glycated albumin. Background technique [0002] Diabetes is a type of metabolic disease that occurs in people with high blood sugar, which can lead to serious damage to most systems of the body, especially nerves and blood vessels. According to the estimates of the World Health Organization, the number of people with diabetes in China ranks first in the world. Diabetes will become the most serious public health problem in China in the next 50 years. [0003] Glycated albumin (Glycatedalbumin, GA) refers to the product formed by the non-enzymatic glycation reaction between glucose and the N-terminal of albumin in human serum, 90% of which react with the l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12Q1/37C12Q1/28C12Q1/26C12R1/685C12R1/68C12R1/66C12R1/645
CPCC12N9/0032C12Y105/03G01N33/6842G01N2333/765G01N2333/90638G01N2333/90672C12Q1/26C12Q1/28C12Q1/37C12Q2326/96
Inventor 邹炳德邹继华汪屹贾江花
Owner NINGBO MEDICAL SYSTEM BIOTECHNOLOGY CO LTD
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