Fucosidase and application of fucosidase to preparation of Bombay phenotype red blood cells

A technology of fucosidase and erythrocytes, applied in the field of glycobiology technology and engineering, can solve problems such as inability to import Bombay-type or Bombay-like individuals, difficulty in finding donors, and unfavorable conditions

Inactive Publication Date: 2020-02-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since most people's blood types are A, B, AB or O, and the population distribution of each blood type is not very different, when a patient urgently needs blood products, each blood transfusion institution will have a certain amount of support for the four blood types. Inventory to ensure the normal supply of blood, even in the case of emergency blood supply, it is relatively easy to find a suitable blood donor, but for patients with Bombay blood type or part of Bombay blood type, the situation is not optimistic, in the case of urgent need for blood products In particular, it is often difficult to find a suitable donor when the demand for red blood cells is high. In addition, in some cases, the human body will produce a relatively high level of autoantibodies to induce autoimmune diseases. Studies have found that using glycan When autoantibodie...

Method used

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  • Fucosidase and application of fucosidase to preparation of Bombay phenotype red blood cells
  • Fucosidase and application of fucosidase to preparation of Bombay phenotype red blood cells
  • Fucosidase and application of fucosidase to preparation of Bombay phenotype red blood cells

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preparation example Construction

[0075] 1.2 Preparation of reaction substrate

[0076] The demonstration of this enzymatic activity was carried out using three types of substrates in the present invention.

[0077] 1. The development substrate pNP-Fuc was purchased from carbosynth company in the UK.

[0078] 2.3-FL, Lewis X and other non-blood group antigen oligosaccharides were purchased from carbosynth company in the UK; blood group antigen oligosaccharides A, B and H types 1, 2, and 4 were purchased from Elicityl OligoTech; 2'-FL is considered to be blood type 5 Antigen substances were purchased from Carbosynth Company, UK.

[0079] 3. Various types of concentrated red blood cells came from the Blood Transfusion Department of Ruijin Hospital and Shanghai Blood Center; the human ABO anti-typing red blood cell kit came from Changchun Broad Biotechnology Co., Ltd. Anti-H reagent is from Shanghai

[0080] The substrate pNP-Fuc and each oligosaccharide were reconstituted with ultrapure water, and 10 mmol / L was...

Embodiment 1

[0095] Example 1: csFase I digestion of pNP-Fuc and its enzymatic properties experiment

[0096] 1. Identification of fucosidase activity

[0097] Preparation of the reaction substrate: the substrate pNP-Fuc lyophilized powder (purchased from carbosynth company) was rethawed with distilled water at a concentration of 10 mM, and stored as a preservation solution.

[0098] Enzyme digestion reaction solution: 25mM phosphate solution (pH 5.0)

[0099] Enzyme digestion system: Substrate pNP-Fuc preservation solution 10 μL, enzyme 1 μL, add phosphate buffer to make up to 60 μL volume. As shown in Table 2 below, number 1 is used as the experimental group, and number 2 is used as the control group.

[0100] Table 2. Configuration of reaction system

[0101]

[0102] Reaction conditions: 37°C, 1h, immediately after the reaction, add 1M Na 2 CO 3 90 μL, stop the reaction, and use a microplate reader to measure OD=405nm. Based on the above results, it was judged whether the enzy...

Embodiment 2

[0109] Example 2: Enzyme digestion experiment of oligosaccharides

[0110] Substrate configuration: All oligosaccharides, including blood group-related and non-blood group antigen oligosaccharides, were prepared with ultrapure water to a concentration of 10mM.

[0111] Reaction system: 8 μL of phosphate buffer solution, 1 μL of oligosaccharide preservation solution, 1 μL of enzyme, a total of 10 μL of reaction system, reacted at 37°C for 1 hour.

[0112] Detection of enzyme digestion reaction: In this example, two methods are used to detect the products of oligosaccharide digestion

[0113] Method 1: detect according to the L-fucose detection kit (megazyme, Ireland). For the specific method, refer to the kit manual. The kit uses fucose dehydrogenase to oxidize fucose, and NADP+ to generate NADPH, NADPH can be measured by the absorbance value at 340nm and quantified according to the calibration curve, so as to quantify the fucose produced by enzymatic cleavage of oligosacchari...

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Abstract

The invention belongs to the field of glycobiology technology and engineering, and particularly relates to novel fucosidase and enzymatic activity and application thereof. The fucosidase can effectively act on polysaccharide structures located on the cell surfaces, and the fucosidase is named as cell surface fucosidase I (csFase I). It is found and proved that csFase I can effectively remove alpha-1,2 fucose on a H-antigen on the cell surfaces of active red blood cells, can effectively convert O-type red blood cells into rare Bombay phenotype red blood cells, and can provide a simple and fastnew way for solving the current situation of extremely difficult blood supply to patients with a Bombay phenotype blood type; and a new method is provided for the research of glycobiology and biomedicine.

Description

technical field [0001] The invention belongs to the field of glycobiology technology and engineering, and relates to the preparation method and application of fucosidase, especially the application in the preparation of Bombay-type red blood cells. Background technique [0002] The prior art discloses that glycosylation modification is a major modification on the cell surface, and plays an extremely important function in the recognition process between cells; as a kind of glycosylation, fucosylation widely exists in various The cell surface also plays an important biological function [1]. [0003] L-fucose is widely found in mammalian, plant and insect cells, and glycoconjugates containing L-fucose play a vital role in many physiological and pathological processes of these organisms, such as inflammatory response, Bacterial and viral infections, tumor metastasis, occurrence of genetic diseases, fertilization process, immune activation effects of antibodies, and allergic rea...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/63C12N5/078
CPCC12N9/2402C12Y302/01063C12N15/63C12N5/0641
Inventor 陈力李天胜
Owner FUDAN UNIV
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