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Modified AFU (a-L-fucosidase) and preparation method thereof, and application of modified AFU in AFU detection

A technology of fucosidase and detection kit, which is applied in the direction of glycosylase, biochemical equipment and methods, and microbial measurement/inspection. Easy operation, wide detection range, and improved stability

Inactive Publication Date: 2019-10-25
上海睿康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, through experimental testing, it is found that there are differences in the anti-interference performance of various reagents on the market, all of which are interfered by bilirubin, and some manufacturers’ reagents are interfered by hemoglobin and chyle
In addition, in terms of sample selection, reagents from some manufacturers are easily interfered by heparin, resulting in high results

Method used

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  • Modified AFU (a-L-fucosidase) and preparation method thereof, and application of modified AFU in AFU detection
  • Modified AFU (a-L-fucosidase) and preparation method thereof, and application of modified AFU in AFU detection
  • Modified AFU (a-L-fucosidase) and preparation method thereof, and application of modified AFU in AFU detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This example provides a modified α-L-fucosidase, wherein 12 amino acids in the N-terminal non-functional amino acid chain of the α-L-fucosidase are excised, and α-L-fucosidase The peptides 101-128 of the a-helix of the glycosidase are modified by methylation, and the peptide chains are cross-linked by disulfide bonds.

[0038] Its preparation method is as follows:

[0039] Use ethylene glycol ether as a solvent, use enolase to hydrolyze α-L-fucosidase, mix enolase and α-L-fucosidase at a mass ratio of 1:3 to 5, and stir Place in a 37°C incubator overnight.

[0040] Add dimethyl sulfate to the α-L-fucosidase solution that has completed the removal of 12 amino acids in the N-terminal non-functional amino acid chain, adjust the pH to 6.0-7.0 with glacial acetic acid, stir overnight at room temperature, and then Add mercaptoethanol and mercaptan and stir for 4-8 hours to obtain.

Embodiment 2

[0042] This example provides a modified α-L-fucosidase, wherein 12 amino acids in the N-terminal non-functional amino acid chain of the α-L-fucosidase are excised, and α-L-fucosidase The peptides 101-128 of the a-helix of the glycosidase are modified by methylation, and the peptide chains are cross-linked by disulfide bonds.

[0043] Its preparation method is as follows:

[0044] Using ethylene glycol ether as a solvent, hydrolyzing α-L-fucosidase with leucine aminopeptidase, mixing enolase and α-L-fucosidase at a mass ratio of 1:3~5 , placed in a 37°C incubator overnight after stirring.

[0045] Add diazomethane to the α-L-fucosidase solution that has completed the removal of 12 amino acids in the N-terminal non-functional amino acid chain, adjust the pH to 6.0-7.0 with glacial acetic acid, stir overnight at room temperature, and then add Mercaptoethanol and mercaptan were stirred for 4-8 hours to obtain.

Embodiment 3

[0047] This example provides a modified α-L-fucosidase, wherein 12 amino acids in the N-terminal non-functional amino acid chain of the α-L-fucosidase are excised, and α-L-fucosidase The peptides 101-128 of the a-helix of the glycosidase are modified by methylation, and the peptide chains are cross-linked by disulfide bonds.

[0048] Its preparation method is as follows:

[0049] Using ethylene glycol ether as a solvent, hydrolyze α-L-fucosidase with trypsin, mix enolase and α-L-fucosidase at a mass ratio of 1:3~5, stir and place 37°C incubator overnight.

[0050] Add methyl chloride to the α-L-fucosidase solution that has completed the removal of 12 amino acids in the N-terminal non-functional amino acid chain, adjust the pH to 6.0-7.0 with glacial acetic acid, stir overnight at room temperature, and then add hydrophobic Base ethanol, mercaptan and stir for 4-8 hours, that is.

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Abstract

The invention provides modified AFU (a-L-fucosidase) and a preparation method thereof, and an application of the modified AFU in AFU detection. In the modified AFU, 12 amino acids in the N-terminal nonfunctional amino acid chain of the AFU are cut off, the 101th-128th peptide fragments of AFU a-spiral are subjected to methylated modification, and peptide chains are crosslinked by a disulfide bond.A stable AFU detection kit prepared by the modified AFU comprises a reagent R and a calibration product, the reagent R includes a buffer solution, the modified AFU, a surfactant, a stabilizer and a corrosion remover; the calibration product includes a buffer solution, a surfactant, the AFU, a stabilizer and a corrosion remover. The reagent kit has the advantages of high stability, high flexibility and wide detection range.

Description

technical field [0001] The invention relates to the technical field of α-L-fucosidase detection, in particular to a modified AFU and its preparation and application in the detection of α-L-fucosidase. Background technique [0002] α-L-fucosidase (α-L-fucosidase, AFU), as a lysosomal acid hydrolase, widely exists in various tissues in the human body, such as liver, kidney, brain, pancreas, and embryonic tissue. It was first discovered in In cell lysosomes, it is mainly involved in the hydrolysis of fucose-containing carbohydrate complexes. Under normal circumstances, AFU is released into the blood as a product with the metabolism of cells. However, when the above-mentioned tissues are pathologically affected, the activity of AFU will increase. In 1977, Bauer et al. observed in animal experiments that the AFU activity in Morris mouse liver cancer tissue was 7 times higher than that in normal liver, and it was related to the growth period of the tumor. After Deugnier et al. f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12Q1/34
CPCC12N9/2402C12Q1/34C12Y302/01051
Inventor 房君江林爱兰
Owner 上海睿康生物科技有限公司
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