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
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[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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