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Novel staphylococcus protein A and preparation method thereof and application

A staphylococcal protein and affinity technology, applied in the biological field, can solve the problems of low purification efficiency and low affinity

Inactive Publication Date: 2017-07-07
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, when using affinity chromatography technology containing protein A, there are problems such as low affinity and low purification efficiency. Therefore, when using this affinity chromatography technology, an improved protein A is needed to achieve higher affinity. Improve purification effect

Method used

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  • Novel staphylococcus protein A and preparation method thereof and application
  • Novel staphylococcus protein A and preparation method thereof and application
  • Novel staphylococcus protein A and preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of recombinant staphylococcal protein A

[0020] 1. Construction of recombinant staphylococcal protein A gene monomer

[0021] By chemical synthesis, design and synthesize a sequence monomer of a repeat fragment of the staphylococcal protein A gene (the sequence of the repeat fragment is shown in the 47th-217th nucleotides shown in SEQ ID NO: 2), which includes a length of 171bp The repeated fragments, and the NcoI restriction site connected to the expression vector, 6 His (for affinity chromatography, combined with nickel column, reducing purification steps), EK restriction site (for His and DDDDK is cut off to form the correct Protein A) and Acll restriction site (AACGTT, used to join multiple repeat fragments). In addition, it also includes the stop codon TAA, and a total of 9 bp of BamH I restriction endonuclease linker for cloning. Labeled "Monomer 1".

[0022] In addition, by means of chemical synthesis, a sequence monomer of a repeat frag...

Embodiment 2

[0036] Example 2 Detection of Recombinant Staphylococcal Protein A and Antibody Binding Activity by ELISA

[0037] ELISA method was used to detect the protein A1 and antibody binding activity prepared in the above-mentioned embodiment 1, and the specific process was as follows:

[0038] 1) Coating: Coat each well of a 96-well plate with 150 μl of Protein A 1 sample, 38°C, 1 h;

[0039] 2) Blocking: each well was blocked with 150 μl of 1% BSA at 38° C. for 1 hour;

[0040] 3) Add primary antibody: Add about 150ug human IgG antibody (purchased from Mlbio) to each well, 38°C, 1h;

[0041] 4) Add secondary antibody: Add about 150 μl, 1:1000 horseradish peroxidase-labeled antibody (purchased from Mlbio Company) to each well, 38°C, 1h;

[0042] 5) Color development.

[0043] The same method was used to detect the binding activity of commercially available Protein A (purchased from Guduo Biotech) and antibody.

[0044] ELISA test results: Protein A 1 of the present invention is com...

Embodiment 3

[0045] Example 3 Preparation of Recombinant Staphylococcal Protein A Sepharose Affinity Chromatography Medium

[0046] Utilize the Protein A1 of embodiment 1 to prepare agarose gel affinity chromatography medium, comprise the following steps:

[0047] 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;

[0048] 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.

[0049] After testing, the characteristics of the prepared recombinant staphylococcal protein A Sepha...

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Abstract

The invention discloses a novel staphylococcus protein A and a preparation method thereof and an application. The novel staphylococcus protein A is characterized in that the amino acid sequence of the novel staphylococcus protein A is shown as SEQ ID NO: 1. The novel staphylococcus protein A of the invention can be coupled with a solid phase carrier to acquire an affinity chromatography medium; the affinity chromatography medium has the advantages of strong protein combination specificity, high affinity, and high purifying efficiency, and can be used for separating and purifying HER-2 antibody.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant protein A and its preparation method and application. Background technique [0002] Staphylococcus aureus protein A (Staphylococcus aureus protein A, SPA) is a cell wall-associated protein of Staphylococcus aureus. Adsorbs IgG and IgG-containing immune complexes. In 1940, Vevwey found that some Staphylococcus aureus contained a substance that could form a precipitate with normal human serum in a two-way diffusion test. In 1959, Jensen also discovered a similar phenomenon and named it the A antigen. In 1960, Grov named it staphylococcal protein A, referred to as SPA protein A. In 1963, Lofkvist et al. isolated the A antigen and proved that it is a protein and is different from sugar. [0003] Protein A (protein A) has the ability to specifically bind to antibodies. Protein A affinity chromatography column has become an affinity column widely used in the fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70C12N1/21G01N33/68C07K1/22
CPCC07K14/31C07K1/22G01N33/68G01N2333/31
Inventor 路玲玉黎健荣李丽君胡湘丽顾云凯
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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