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HEMOGLOBIN A1c MEASUREMENT METHOD AND MEASUREMENT KIT

A technology of hemoglobin and determination method, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, measurement devices, etc., and can solve the problem of not finding amadoriase and the like

Pending Publication Date: 2016-06-15
KIKKOMAN CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Based on the above reasons, amadoriase that directly oxidizes the β chain of HbA1c to generate hydrogen peroxide and an HbA1c measurement method using it have been sought, but no amadoriase showing such enzymatic activity has been found so far. madoriase

Method used

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  • HEMOGLOBIN A1c MEASUREMENT METHOD AND MEASUREMENT KIT
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  • HEMOGLOBIN A1c MEASUREMENT METHOD AND MEASUREMENT KIT

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0659] (1) Preparation of recombinant plasmid pKK223-3-CFP-T7DNA

[0660] Escherichia coli JM109 (pKK223-3-CFP-T7) strain (refer to International Publication No. 2007 / 125779) having a recombinant plasmid of the Amadoriase gene (sequence number 2) from the genus Conechaeta was inoculated in 3 ml of LB-amp medium [1% (w / v) bacto-tryptone, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, 50 μg / ml ampicillin], shaking at 37°C for 16 hours , to obtain cultures.

[0661]The culture was collected by centrifugation at 10,000×g for 1 minute to obtain bacterial cells. From this bacterial cell, recombinant plasmid pKK223-3-CFP-T7 was extracted and purified using GenElutePlasmid Miniprep Kit (manufactured by Sigma-Aldrich) to obtain 2.5 μg of recombinant plasmid pKK223-3-CFP-T7 DNA.

[0662] (2) Site-specific modification of recombinant plasmid pKK223-3-CFP-T7DNA

[0663] Using the obtained recombinant plasmid pKK223-3-CFP-T7 DNA as a template, a PCR reaction was performed under the following cond...

Embodiment 2

[0740] (Production and purification of various Amadoriases)

[0741] (Production and Purification of Modified Amadoriase from Conechaeta)

[0742] Escherichia coli JM109 (pKK223-3-CFP-T7), Escherichia coli JM109 (pKK223-3-CFP -T7-62D), Escherichia coli JM109 (pKK223-3-CFP-T7-H20), and Escherichia coli JM109 (pKK223-3-CFP-T7-H21), and Escherichia coli JM109 (pKK223-3-CFP-T7- H35), the bacteria were planted in 120 ml of LB-amp medium to which IPTG was added to a final concentration of 0.1 mM, and cultured at 25° C. for 16 hours. After each cultured cell obtained was washed with 10 mM potassium phosphate buffer (pH 7.0), the cell was suspended in the same buffer, ultrasonically disrupted, and centrifuged at 20,000×g for 10 minutes to prepare 24 ml of a crude enzyme solution.

[0743] The prepared crude enzyme solution was adsorbed on a solution containing 1.35M (NH 4 ) 2 SO 4 12ml of Butyl Toyopearl 650C resin (manufactured by TOSOH) equilibrated with 10mM potassium phosphat...

Embodiment 3

[0774] (Introduction of point mutations into various amadoriases)

[0775] The reactivity of the Amadoriase derived from the genus Conechaeta to αF6P was increased by introducing the above mutation. Therefore, by introducing the same mutation at the corresponding position in the amino acid sequence of amadoriase derived from other biological species, the same response to αF6P can be expected using the information obtained by known alignment processing based on sequence homology as a reference. Sex rises. Therefore, in fact, mutations were also introduced at the corresponding positions for a plurality of amadoriases other than the Amadoriase derived from the genus Trichochaeta.

[0776] 1. Introducing a point mutation into the fructosyl peptide oxidase gene from Penicillium terreus

[0777] SEQ ID NO: 40 is the amino acid sequence of fructosyl peptide oxidase (hereinafter referred to as EFP-T5) from Penicillium terreus, through the recombinant plasmid pUTE100K'- Escherichia ...

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Abstract

Provided is an amadoriase characterized in acting on the beta-chains of hemoglobin A1c (HbA1c) to generate hydrogen peroxide. A HbA1c measurement method and measurement reagent kit using same are also provided. A HbA1c measurement method uses an amadoriase characterized in having a specific activity of 0.1 U / mg or more with respect to alphaF6P and in oxidizing the amino terminus of the beta-chains of HbA1c to generate hydrogen peroxide, and a measurement reagent kit comprises said amadoriase. The present invention provides a measurement method and measurement kit that are able to quantify HbA1c rapidly, easily and with good precision.

Description

technical field [0001] The present invention relates to a method for measuring hemoglobin A1c in a sample and a kit for measurement used in the method. Background technique [0002] Glycated protein is produced by non-enzymatically forming a covalent bond between the aldehyde group of an aldose such as glucose (a monosaccharide with potential aldehyde group and its derivatives) and the amino group of the protein and undergoing Amadori rearrangement. The amino group of a protein includes an α-amino group at an amino terminal and an ε amino group in a side chain of a lysine residue in a protein. Glycated hemoglobin in which hemoglobin in blood is glycosylated, glycated albumin in which albumin is glycosylated, etc. are known as a glycated protein produced in vivo. [0003] Among these glycated proteins produced in vivo, in the field of clinical diagnosis of diabetes, hemoglobin A1c (HbA1c) has attracted attention as an important blood sugar control marker for diagnosis and sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/06C12Q1/26C12Q1/28
CPCC12N9/0032C12Y105/03G01N33/725C12Y111/01C12Q1/26C12Q1/28G01N21/78G01N2333/805G01N2333/90672G01N2333/908
Inventor 钺阳介一柳敦
Owner KIKKOMAN CORP
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