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Affinity chromatography medium, and preparation method and application thereof

A chromatography medium and affinity technology, applied in the field of affinity chromatography medium and its preparation, can solve the problems of low purification efficiency and low affinity, and achieve the effects of high purification efficiency, high affinity and improved dynamic adsorption capacity

Inactive Publication Date: 2016-07-20
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are problems such as low affinity and low purification efficiency when using the affinity chromatography medium prepared from natural Protein A.

Method used

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  • Affinity chromatography medium, and preparation method and application thereof
  • Affinity chromatography medium, and preparation method and application thereof
  • Affinity chromatography medium, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0026] The present invention is further described below in conjunction with the examples, and the following examples should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, to insert protein-encoding genes into such vectors and plasmids, or to introduce plasmids into host cells, as well as classical cell fusion and Monoclonal antibody screening and purification methods, etc. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Coldspring Harbor Laboratory Press. Preparation of Example 1 Recombinant Staphylococcal Protein A

[0027] 1. Construction of recombinant staphylococcal protein A gene monomer

[0028] By chemical synthesis, design and synthesize a sequence monomer of a r...

Embodiment 2E

[0042] Embodiment 2 ELISA method detects recombinant Staphylococcus protein A and antibody binding activity

[0043] ELISA method was used to detect the proteinA1 and antibody binding activity prepared in the above-mentioned embodiment 1, and the specific process was as follows:

[0044] 1) Coating: Coat each well of a 96-well plate with 100 μl of ProteinA1 sample, 37°C, 1 h;

[0045] 2) Blocking: each well was blocked with 100 μl of 1% BSA at 37° C. for 1 hour;

[0046] 3) Add primary antibody: Add about 100ug of human IgG antibody (purchased from Mlbio) to each well, 37°C, 1h;

[0047] 4) Add secondary antibody: Add about 100 μl, 1:1000 horseradish peroxidase-labeled antibody (purchased from Mlbio) to each well, 37°C, 1h;

[0048] 5) Color development.

[0049] The same method was used to detect the binding activity of commercially available Protein A (purchased from Guduo Biotechnology) and antibody.

[0050] ELISA test results: ProteinA1 of the present invention is com...

Embodiment 3

[0051] Embodiment 3 Preparation of affinity chromatography medium

[0052] Utilize the ProteinA1 of embodiment 1 to prepare agarose gel affinity chromatography medium, comprise the following steps:

[0053] 1. Activation of agarose gel: react in aqueous medium with epichlorohydrin, sodium hydroxide and agarose (5% cross-linked agarose gel), and react 2- After 3 hours, after the reaction, wash with distilled water until neutral and then drain to obtain activated agarose gel;

[0054] 2. Chemical coupling of recombinant staphylococcal protein A to activated agarose gel: react the activated agarose gel obtained in the above step 1 with the Protein A1 prepared in Example 1 at a temperature of 5-25° C. for 15-20 hours, After the reaction, it was washed and dried to obtain staphylococcal protein A sepharose affinity chromatography medium.

[0055] Use the same method to prepare Sephadex affinity chromatography media:

[0056] 1. React with epichlorohydrin, sodium hydroxide and de...

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Abstract

The invention discloses an affinity chromatography medium, and a preparation method and an application thereof. The affinity chromatography medium provided by the invention comprises a solid phase carrier and recombinant staphylococcal protein A coupled with the solid phase carrier. The amino acid sequence of the recombinant staphylococcal protein A is represented by SEQ ID NO:1. The affinity chromatography medium provided by the invention has the advantages of high protein binding specificity, high affinity and high purification efficiency. The affinity chromatography medium can be used for separation and purification of protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an affinity chromatography medium and its preparation method and application. Background technique [0002] With the development of biotechnology, it is possible to prepare protein drugs by recombinant technology. At present, people clone the gene encoding the antibody and transform the gene to be expressed into a suitable expression host cell for cultivation and purification to finally obtain the desired antibody drug. Among them, in the preparation process of antibody drugs, the purification of antibody proteins is the most critical link. According to statistics, more than 60% of the funds in the development of antibody drugs are spent on the downstream purification process. Therefore, the efficiency and cost of the purification process will increase. directly affects the success of drug development. [0003] The most commonly used technique for protein purification is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/285B01J20/30B01D15/38C07K1/22C07K16/00
Inventor 黎健荣路玲玉胡湘丽
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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