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Low-irritation lectin magnetic bead and preparation method of recombinant lectin magnetic bead

A lectin and stimulant technology, which is applied in the field of preparation of lectin magnetic beads and recombinant lectin magnetic beads, can solve the problems of large cell pressure and damage, small cell load capacity of ConA magnetic beads, and easy hardening, etc., to achieve cell pressure Small, low cost, simple preparation effect

Pending Publication Date: 2022-01-11
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ConA magnetic beads prepared by this method have a small cell loading capacity, great pressure and damage to the cells, and are easy to harden, which has a great impact on the downstream experiments of cell capture.

Method used

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  • Low-irritation lectin magnetic bead and preparation method of recombinant lectin magnetic bead
  • Low-irritation lectin magnetic bead and preparation method of recombinant lectin magnetic bead
  • Low-irritation lectin magnetic bead and preparation method of recombinant lectin magnetic bead

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Cell Stimulation Test of Different Lectins.

[0033] In this example, we tested the stress caused by different lectin treatments on the cells. The lectins we picked were concanavalin A, Dictyospora aurantium agglutinin, Gardner seed agglutinin, lentil agglutinin, tomato There are 9 kinds of lectins including lectin, ricin, wheat germ, peanut lectin and soybean lectin, and the expression change of gene spectrum after treatment is used as a detection index in response to cell stress. The specific implementation method is as follows:

[0034] Take 100-1,000,000 293F cells and CHO cells in good condition, wash them twice with washing buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1x Protease inhibitors cocktail), and suspend into 100 ul wash buffer. Add 100 ng of concanavalin A, Dictyospermia aurantium agglutinin, Gardner seed agglutinin, lentil bean agglutinin, tomato agglutinin, ricin, wheat germ, peanut agglutinin and soybean agglutinin (both ...

Embodiment 2

[0036] Example 2: Cell stimulation test of different kinds of magnetic beads.

[0037] In this example, we tested the irritation caused by the treatment of different types of agglutinated magnetic beads on the cells. The magnetic beads we picked included streptavidin magnetic beads (ThermoFisher, M-270, M-280, MyOne-C1 , MyOne-T1), SNAP-Tag magnetic beads (NEB), Flag magnetic beads (Sigma), Halo-Tag magnetic beads (Promega), SpyT magnetic beads (see Hatlem D, Trunk T, et al. International Journal of Molecular Sciences, 2019, 20(9)), NHS-activated magnetic beads (ThermoFisher), and the expression changes of gene profiles after treatment are used as detection indicators in response to cellular stress. The specific implementation methods are as follows:

[0038]Take 100-1,000,000 293F cells and CHO cells in good condition, wash them twice with washing buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1x Protease inhibitors cocktail), and suspend into 100 ul wash buff...

Embodiment 3

[0040] Example 3: Testing of cell stimulation and capture efficiency of different kinds of lectin streptavidin magnetic beads.

[0041] In this example, we compared the stimulation of cells treated with different kinds of lectin streptavidin magnetic beads. The tested lectins were purchased from VECTOR. The specific process is as follows:

[0042] Take 100 ng of lectin protein and suspend it in 100 ul of washing buffer. Take 10 ul streptavidin magnetic beads, wash twice with 100 ul washing buffer, and resuspend in 10 ul washing buffer. Add the magnetic beads to the protein, mix well, and incubate with rotation for 30 min. Wash the magnetic beads twice with 100 ul activation buffer (20 mM HEPES-KOH (pH 7.5), 10 mM potassium chloride, 1 mM calcium chloride, 1 mM manganese chloride), and resuspend in 10 ul activation buffer . Add the magnetic beads to the cells, mix well, rotate and incubate at room temperature for 10 min, place on the magnetic stand to take the supernatant,...

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Abstract

The invention provides a low-irritation lectin magnetic bead, which is composed of a magnetic bead and lectin protein which is covalently bound with the magnetic bead and is provided with labels, the magnetic bead is an SNAP-Tag magnetic bead and a Halo-Tag magnetic bead, the labels are SNAP label and Halo label which can be correspondingly coupled to the magnetic bead, and the lectin protein is concanavalin A and lentil lectin. According to the invention, lectin protein with a specific protein label is covalently bound to a relative capture magnetic bead, and the prepared magnetic bead has the advantages of simple preparation, low cost, small cell pressure, strong stability, lasting binding capacity, large cell bearing capacity, small loss, difficult hardening and the like, and is very suitable for experimental requirements of cell capture.

Description

technical field [0001] The patent of the present invention relates to a preparation method of low-irritant lectin magnetic beads and recombinant lectin magnetic beads, belonging to the field of biotechnology. Background technique [0002] Lectins are a class of proteins that can bind polysaccharide groups and are mainly distributed in the extracellular matrix. A large number of glycoproteins and polysaccharide groups are distributed in the extracellular matrix, and lectins can mediate the stable connection between cells by binding polysaccharide groups. Plant-derived lectins, such as Conconvalina (ConA), Wheat germagglutinin (WGA), Peanut agglutinin (PNA) and soybean lectin (Soybeanagglutinin, SBA), can efficiently bind polysaccharide groups. Therefore, lectins have become important biological tools for capturing glycoproteins. [0003] In recent years, cell separation and capture technology has become the core of many biological technologies, including CUT&RUN, CUT&Tag, ...

Claims

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Application Information

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IPC IPC(8): H01F1/42C12N15/70C12N15/62C07K19/00
CPCH01F1/42C12N15/70C07K14/42C07K2319/20
Inventor 宋东亮侯策刘倩孙睿王嫚江翱陈晶晶曹振
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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