Purified glass bead as well as preparation method and application thereof

A glass microbead and avidin technology, applied in the field of biochemistry, can solve the problems such as the inability to visually observe the binding situation of the target and the difficulty of intuitive observation, and achieve the effect of realizing visualization, facilitating process monitoring, and avoiding high retention ratio.

Pending Publication Date: 2022-06-21
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When purifying the purification medium, it is impossible to visually observe the binding of the target substance; due to the dark color of the microsphere itself, it is difficult to visually observe even when the fluorescent marker is purified

Method used

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  • Purified glass bead as well as preparation method and application thereof
  • Purified glass bead as well as preparation method and application thereof
  • Purified glass bead as well as preparation method and application thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0448] A preferred embodiment of the preparation method of the purified glass microspheres comprises the following steps:

[0449] Step 1: Provide glass microspheres. The glass microspheres are white. The glass microspheres may or may not contain cavities. In one of the preferred modes, the glass microspheres are hollow glass beads.

[0450] Step 2: modifying the silica coating on the surface of the glass microspheres to obtain silica-wrapped glass microspheres, which are the glass microsphere bodies.

[0451] One of the preferred ways is to add glass microspheres into a mixed solvent of ethanol and water and stir, add ammonia water, and dropwise add an ethanol solution of tetraethyl orthosilicate to obtain glass microspheres wrapped in silicon dioxide.

[0452] Step 3: modifying the surface of the glass bead with a carbon-carbon double bond to obtain glass microbeads modified with a carbon-carbon double bond.

[0453] This step can be a one-step method or a step-by-step m...

specific Embodiment approach

[0517] A specific embodiment of preparing the purified glass microbeads with biotin-modified glass microbeads is as follows.

[0518] Specifically, taking an acrylic polymer as an example to provide a linear main chain and a large number of branched chains, the present invention provides the following specific implementation method: use silica-wrapped glass beads as the glass bead body; first use coupling agent 3 -Aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent and also a silane coupling agent, more specifically an aminated silane coupling agent KH550) Chemically modify the silica-wrapped glass beads, introduce amino groups to the outer surface of the glass beads, and complete the SiO 2 Activation modification to obtain amino-modified glass microspheres; then use the covalent reaction between carboxyl and amino groups to covalently couple immobilized molecules (acrylic acid molecules, providing a carbon-carbon double bond and a reactive group carbo...

Embodiment 1

[0596] The DNA template uses a nucleic acid template encoding 8His-mEGFP. Using PCR amplification and homologous fragment recombination methods, mEGFP containing 8His-mEGFP (histidine tag tagged mEGFP, wherein mEGFP is the A206K mutant of eGFP, the nucleotide sequence is shown in SEQ ID No.: 1, the amino acid sequence The DNA fragment encoding the gene as shown in SEQ ID No.: 2) was inserted into a plasmid vector to construct a plasmid vector expressing mEGFP. The plasmid was confirmed to be correct by gene sequencing. The constituent elements of the plasmid include T7 promoter, 5'UTR, leader peptide coding sequence (leader peptide), 8×His (histidine tag), mEGFP coding sequence, 3'UTR, LAC4 terminator, f1 ori (replication initiation site), AmpR promoter, AmpR gene (ampicillin resistance gene), ori (high copy number replication initiation site), lacI promoter, lacI (lac repressor) coding gene and other functional elements . By the RCA amplification method, the plasmid DNA en...

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Abstract

The invention discloses a purified glass bead as well as a preparation method and application thereof. The purified glass bead comprises a white glass bead body, and the outer surface of the purified glass bead is combined with a purification medium or can be combined with the purification medium so as to be used for purifying a fluorescently-labeled target object. The color of the glass beads combined with the fluorescently-labeled target object can be observed, the combination condition of the target object can be visually observed, the visualization of the purification process is realized, and the process is convenient to monitor. A large number of purification media can be further combined through a comb-shaped polymer, the comb-shaped polymer can provide a flexible main chain and can also provide a large number of branch chains, protein combination and elution are facilitated, and the number of the purification media can be greatly increased.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a purified glass microsphere and its preparation method and application. Background technique [0002] The separation and purification of protein is an important downstream link in the production process of biopharmaceuticals. The effect and efficiency of separation and purification directly affect the quality and production cost of protein drugs. For protein purification, materials such as agarose gel are commonly used as purification columns or purification microsphere carriers at this stage. In the prior art, for the separation and purification of antibody substances, production technicians mainly use protein A affinity adsorption columns to separate and purify antibody molecules in fermentation broth or reaction fluid. In the protein A column, the protein A immobilized on the carrier is specifically bound to the specific site of the Fc end of the antibody mo...

Claims

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Application Information

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IPC IPC(8): C08F292/00C08F220/06C08F8/42C08F8/32C07K1/22G01N21/33G01N21/64
CPCC08F292/00C08F8/42C08F8/32C07K1/22G01N21/6428G01N21/33C08F220/06
Inventor 郭敏吴亮于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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