Unlock instant, AI-driven research and patent intelligence for your innovation.

Preparation method and application method of lysozyme affinity filler

An application method and lysozyme technology, which are applied in the preparation methods of peptides, chemical instruments and methods, biochemical equipment and methods, etc., to achieve the effects of increasing solubility, increasing expression, and low cost of ligands

Pending Publication Date: 2021-06-29
CHANGZHOU SMART LIFESCI CO LTD
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the separation methods commonly used for lysozyme antibody include crystallization method, precipitation method, ion exchange method, affinity chromatography, etc. Among them, the affinity chromatography technology has the advantages of high yield, high purity, and the ability to maintain the natural state of biological macromolecules. Affinity fillers such as protein A and protein G can be used for the separation of conventional recombinant antibodies. For example, patent CN109336968A discloses a method for purifying antibodies by protein A affinity chromatography, and the following protein A affinity chromatography columns are recycled to purify target antibodies: The filler is MabSelect LX, Amsphere A3 or Praesto Jetted A50, the column height is 5-15cm, and the linear flow rate is 200-500cm / h; before eluting the target antibody with the eluent, use a pH value of 5-6, conductivity The column bed is washed with a buffer solution of 20-100 mS / cm. This patent not only reduces the cost of the medium, but also improves the purification efficiency by selecting a packing material with high loading capacity, good mass transfer capacity, and fast operating flow rate; but the method mainly uses Combined with antibody Fc fragments for purification, it is impossible to separate and purify antibodies without Fc fragments such as single chain antibodies or nanobodies

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application method of lysozyme affinity filler
  • Preparation method and application method of lysozyme affinity filler
  • Preparation method and application method of lysozyme affinity filler

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] It should be noted that the activated solid phase carrier NHS-Activated Beads 4FF in this example was produced by Changzhou Tiandi Renhe Biotechnology Co., Ltd.

[0041] The invention discloses a preparation method of lysozyme affinity filler (through carboxyl coupling on the surface of microspheres), comprising the following steps:

[0042] (1) Wash the NHS-Activated Beads 4FF three times with 1mM HCl solution, and then wash with 0.2M NaHCO at pH 8 3 Wash once with a mixed solution of 0.5M NaCl;

[0043] (2) Add the same volume of lysozyme solution to the cleaned NHS-Activated Beads 4FF, shake and react at 28°C for 4 hours, collect the coupling packing after the reaction, wash with ion water, add 2 times the column volume of 1M ethanolamine, Shaking reaction at 28°C for 1h;

[0044] (3) Take out the reaction system in step (2), drain the ethanolamine, wash the filler once with 3 times the column volume of deionized water, and then use a mixed solution of 0.1M acetic ...

Embodiment 2

[0056] The invention discloses a preparation method of lysozyme affinity filler (through hydroxyl coupling on the surface of microspheres), comprising the following steps:

[0057] (1) Filler activation: add 90ml of 1M NaOH to 100ml of agarose gel solution, adjust its pH to 14, then add 15ml of epichlorohydrin and 200mg of sodium borohydride, react at 40°C for 2 hours, and use Ionized water cleaning, collecting filler;

[0058] (2) Ligand coupling: add 100ml, 10mg / ml lysozyme to the reaction system in step (1), and then add 0.1M Na 2 CO 3 , adjust the pH of the reaction system to 9, react at 25°C for 24 hours, wash with deionized water after completion, and collect the filler;

[0059] (3) Blocking reaction: Add 100ml of 1M ethanolamine to the reaction system in step (2), adjust the pH of the reaction system to 9, react at 40°C for 1 hour, and wash with deionized water after the end to obtain lysozyme affinity filler.

[0060] The invention also discloses a method for appl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a preparation method and an application method of a lysozyme affinity filler. The preparation method is characterized in that lysozyme is fixed on a solid-phase carrier by bonding with hydroxyl, carboxyl, amino or epoxy groups on the surface of the solid-phase carrier, thereby obtaining the lysozyme affinity filler. When the affinity lysozyme filler is used for separating lysozyme antibody fusion proteins, microbial metabolites and lysozyme antibodies obtained through other ways, the separation efficiency is high, and the purity of the obtained lysozyme antibodies reaches up to 95% or above.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and pharmaceutical engineering, and in particular relates to a preparation and application method of lysozyme affinity filler. Background technique [0002] Lysozyme, also known as muramidase or N-acetylmuramic glycan hydrolase, mainly destroys the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, Decompose the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, causing the contents of the cell wall to rupture and the bacteria to rupture. Lysozyme can also directly combine with negatively charged viruses, and form double salts with DNA, RNA, and deradiated proteins to inactivate viruses. Therefore, lysozyme has antibacterial, anti-inflammatory, antiviral and other effects. [0003] Lysozyme is an important part of the endogenous immune system in various animal tissues. Different tissues have different normal levels; changes ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/12C12N11/10C12N11/14C12N11/089C07K16/40C07K1/22
CPCC12N11/10C12N11/12C12N11/08C12N11/14C12N9/2462C12Y302/01017C07K16/40C07K1/22
Inventor 单玉飞曹飞婷方利
Owner CHANGZHOU SMART LIFESCI CO LTD