Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof

A technology of magnetic nanoparticles and sialic acid oligosaccharides, which is applied in the field of sialic acid oligosaccharides-magnetic nanoparticles and its preparation, can solve the problems of difficult preservation and application, high price, variability, etc., and facilitate preservation and application, and preparation Simple and convenient, good practical effect

Active Publication Date: 2017-04-26
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antibodies are biological products, expensive and easily denatured, so the prepared antibody-magnetic nanoparticles are expensive and difficult to store and apply, let alone repeated recycling
Moreover, due to the generally high specificity of antibodies, the magnetic nanoparticles coated with each antibody can only enrich and isolate specific influenza virus strains, which is lacking in wide applicability.

Method used

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  • Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof
  • Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof
  • Sialyloligosaccharide-magnetic nanoparticles as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0090] 1. Preparation of sialooligosaccharide propargyl glycoside shown in formula X

[0091]

[0092] Get compound 3 (final concentration is 10mM) and 197.5 mg CMP-N-acetylneuraminic acid (CMP-Neu5Ac, final concentration is 15mM) after dissolving in 20 milliliters of Tris-HCl (pH 8.0) buffer solution, the reaction system The temperature was raised to 37°C, and then 500 microliters of Pm2,3-ST (1U) was added. After 2-3 hours, the thin layer chromatography (TLC) detected that the raw material had completely disappeared, and the reaction solution was passed through an ultrafiltration membrane to remove the enzyme protein, and then frozen After drying and concentrating, the compound of formula X is obtained after purification by gel column G-15.

[0093] 2. Preparation of sialooligosaccharide propargyl glycoside shown in formula XI

[0094]

[0095] Get compound 4 (final concentration is 10mM) and 197.5 milligrams of CMP-N-acetylneuramine (CMP-Neu5Ac, final concentration i...

Embodiment 1

[0096] Embodiment 1, the preparation of sialooligosaccharide-magnetic nanoparticles

[0097]

[0098] 1. Magnetic nanoparticles (Fe 3 o 4 ) preparation

[0099] Add polystyrenesulfonic acid-maleic acid copolymer sodium salt (PSSMA 3:1, 1g) into 20mL ethylene glycol and stir to dissolve, then add ferric chloride hexahydrate (0.54g), anhydrous sodium acetate (1g) , and stir until completely dissolved. Then the above solution was transferred to a 25 ml polytetrafluoroethylene-lined high-pressure hydrothermal reaction kettle, and the reaction kettle was placed in an oven preheated to 200° C. for heating and reaction for 10 hours. The reaction kettle was taken out and cooled to room temperature naturally, the magnetic microspheres were separated by magnetic separation, washed with pure water to remove unreacted reactants and by-products, dried in vacuum, and superparamagnetic ferric oxide microspheres (Fe 3 o 4 ).

[0100] 2. Fe 3 o 4 Preparation of @PMAA

[0101] Ferro...

Embodiment 2

[0114] Example 2. Enrichment and separation of influenza virus HA protein by sialic acid oligosaccharides-magnetic nanoparticles, and testing its host specificity

[0115] 1. Sample preparation:

[0116] Take 125 μl of the sialooligosaccharide-magnetic nanoparticle PBST solution (1 mg / ml) obtained in Example 1 and place in each 1.5ml centrifuge tube, separate with a magnetic separation rack, and then wash twice with 200 μl PBS;

[0117] Block with 200 μl 3% BSA (dissolved in PBS), shake at 30°C for 1.5h;

[0118] Dissolve in 100 μl 1% BSA, add 1 μg HA protein (influenza virus vieH5HA protein or influenza virus 09H1HA protein), mix well, incubate at 4°C for 1 hour, separate with magnetic separation rack, wash twice with 200 μl PBST (0.05% Tween-20);

[0119] Add 40 μl PBS and 10 μl 5× protein loading buffer, mix well, boil for 10 minutes, separate with a magnetic separation rack, and save the supernatant for later use.

[0120] 2. Electrophoresis:

[0121] Prepare 10% separa...

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Abstract

The invention discloses sialyloligosaccharide-magnetic nanoparticles as well as a preparation method and an application thereof. The general structural formula of the sialyloligosaccharide-magnetic nanoparticles is shown in formula X-1 or X-2. The sialyloligosaccharide-magnetic nanoparticles are prepared from alpha 2,3 sialyloligosaccharide and alpha 2,6 sialyloligosaccharide which are specifically bound with influenza virus HA protein by joining to magnetic nanoparticles through a covalent bond, the HA protein of type A influenza viruses of subtypes such as H1 and H5 and type A influenza virus strains of subtypes such as H1, H3, H5 and H7 are enriched and separated, and the receptor specificity of the influenza viruses is analyzed and detected. Accordingly, human influenza viruses and avian influenza viruses are selectively enriched, separated and purified, enrichment and separation or analysis of receptor specificity can be performed on samples with lower concentration and complex composition, and the sialyloligosaccharide-magnetic nanoparticles have quite good practicability and are of great significance in prevention and control of influenza viruses.

Description

technical field [0001] The invention belongs to the field of biological materials, and relates to a sialic acid oligosaccharide-magnetic nano particle and a preparation method and application thereof. Background technique [0002] Influenza (flu) is a zoonotic infectious disease caused by influenza virus, and its host involves multiple animals such as people, pigs, birds, horses and dolphins. Sensitive and reliable influenza virus detection methods are becoming more and more important. But so far, the most standardized influenza virus isolation and identification process involves the use of cells, chicken embryos or experimental animals to amplify the virus, and then conduct a series of experimental isolation and identification. The whole process takes at least two weeks, which is time-consuming and labor-intensive. And the actual sample to be tested is generally a relatively complex biological sample with an extremely low content of influenza virus. Other widely used infl...

Claims

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Application Information

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IPC IPC(8): C07H15/10C07H1/00C08B37/00H01F1/42C12N7/00C07K14/11G01N33/569
CPCC07B2200/11C07H1/00C07H15/10C07K14/005C08B37/00C12N7/00C12N2760/16151G01N33/56983H01F1/42
Inventor 李学兵张振兴
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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