Staphylococcal protein A, preparation method thereof and use thereof

A staphylococcal protein and affinity technology, applied in the biological field, can solve the problems of low affinity and low purification efficiency, and achieve the effects of high affinity, strong protein binding specificity, and improved dynamic adsorption capacity

Inactive Publication Date: 2012-07-11
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

[0004] However, when using the affinity chromatography technology containing staphylococcal protein A, there are problems such as low affinity and low purification efficiency. Therefore, when using the affinity chromatography technology, an improved staphylococcal protein A is needed to achieve more High affinity, improve the effect of purification

Method used

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  • Staphylococcal protein A, preparation method thereof and use thereof
  • Staphylococcal protein A, preparation method thereof and use thereof
  • Staphylococcal protein A, preparation method thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 , Synthesis of staphylococcal protein A gene monomer

[0026] Through chemical synthesis, the sequence of a repetitive fragment of staphylococcal protein A gene was designed and synthesized. The sequence includes a length of 174bp, a NcoI restriction site connected to the expression vector, and 6 His (for affinity chromatography, Combine with nickel column to reduce purification steps), EK restriction site (used to cut off His and DDDDK to form the correct Protein A) and AccI restriction site (used to join multiple repetitive fragments). In addition, it also includes the stop codon TAA and the BamH I restriction endonuclease linker for cloning, a total of 9bp.

[0027] Through chemical synthesis, the sequence of a repetitive fragment of the staphylococcal protein A gene was designed and synthesized. The sequence included a length of 174 bp with AccI restriction sites at both ends to construct a sequence containing multiple repetitive fragments.

[0028] The sequenc...

Embodiment 2

[0029] Example 2 , Construction of a pET32a vector containing a staphylococcal protein A gene monomer

[0030] The above-mentioned staphylococcal protein A gene monomer was excised with restriction endonucleases NcoI and BamH I, and connected to the vector pET32a digested with NcoI and BamH I, transformed into E. coli, and screened for ampicillin-resistant transformants. Plasmid extraction, restriction enzyme digestion and identification proved that the staphylococcal protein A gene monomer has been cloned into pET32a.

Embodiment 3

[0031] Example 3 , Construction of pET32a-P vector containing staphylococcal protein A gene

[0032] The above-mentioned pET32a vector containing a staphylococcal protein A gene monomer and staphylococcal protein A gene monomer were digested with AccI, and the corresponding fragments were recovered and ligated, transformed into Escherichia coli, selected transformants with ampicillin resistance, and extracted by plasmid The pET32a-P vector of Staphylococcal protein A containing 3 protein A gene monomers was obtained by restriction digestion and screening. The gene sequence of the pET32a-P vector is as shown in SEQ ID No: 2 after detection.

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Abstract

The invention discloses a staphylococcal protein A, a nucleotide sequence coding the staphylococcal protein A, and an affine chromatography media comprising the staphylococcal protein A and a chromatography media carrier. The amino acid sequence of the staphylococcal protein A provided in the invention is represented by SEQ ID NO:1, 5, 7 or 11. The affine chromatography media prepared by using the staphylococcal protein A provided in the invention has the advantages of strong protein combination specificity, high affinity and substantial improvement of the purification efficiency, and the dynamic adsorption load of the affine chromatography media is obviously higher than dynamic adsorption loads of Protein A Sepharose 4Fast Flow sold in the market.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a staphylococcal protein A and a preparation method and application thereof. Background technique [0002] Staphylococal Protein A (Staphylococal Protein A, SPA) is a protein isolated from the cell wall of Staphylococcus aureus. In 1940, Vevwey discovered that certain 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 A antigen; in 1960 Grov named it Staphylococcal protein A, or SPA protein A for short. In 1963, Lofkvist and others isolated A antigen and proved it to be a protein. , And is different from sugar. [0003] Staphylococcal protein A is an important medium for antibody purification. With the rapid development of my country's biotechnology industry, especially the biopharmaceutical industry represented by monoclonal antibody dr...

Claims

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Application Information

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IPC IPC(8): C07K14/31C12N15/31C12N15/63C12N15/70G01N33/53C07K16/00B01J20/285
Inventor 郭亚军王进秋倪华胡辉
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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