Novel ProteinA, and preparation method and use of novel ProteinA

An affinity, staphylococcal protein technology, applied in the field of ProteinA and its preparation, to achieve the effects of strong protein binding specificity, improved purification efficiency, and increased 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

With the rapid development of the biotechnology industry, especially the biopharmaceutical industry represented by monoclonal antibody drugs, the market demand for staphylococcal protein A is increasing rapidly, and the requirements for the affinity and purification efficiency of ProteinA affinity columns are getting higher and higher , the existing ProteinA affinity column can not fully meet the requirements, therefore, the development of a ProteinA with higher affinity is still urgently needed

Method used

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  • Novel ProteinA, and preparation method and use of novel ProteinA
  • Novel ProteinA, and preparation method and use of novel ProteinA
  • Novel ProteinA, and preparation method and use of novel ProteinA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Staphylococcus protein A gene monomer preparation

[0019] 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 46th-210th nucleotides shown in SEQ ID NO: 2), which includes a repeat length of 165bp fragment, and the NcoI restriction site connected to the expression vector, 6 His (used for affinity chromatography, combined with nickel column, reducing purification steps), EK restriction site (used to cut His and DDDDK Go, form the correct ProteinA) and AccI restriction site (gtagac, used to join multiple repeat fragments). In addition, it also includes the stop codon TAA, and a BamHI restriction endonuclease linker for cloning with a total of 9 bp. Labeled "Monomer 1".

[0020] In addition, by means of chemical synthesis, a sequence monomer of a repeat fragment of the staphylococcal protein A gene (the sequence of the repeat fragment i...

Embodiment 2

[0021] Embodiment 2, construct the pET32a vector containing a staphylococcal protein A gene monomer

[0022] Monomer 1 obtained in Example 1 was digested with restriction endonucleases NcoI and BamHI, then connected to the vector pET32a digested with NcoI and BamHI, transformed into E. After extraction and enzyme digestion identification, it was proved that the staphylococcal protein A gene monomer had been cloned into pET32a, thereby successfully constructing a pET32a vector containing a staphylococcal protein A gene monomer.

Embodiment 3

[0023] Embodiment 3, the construction of the pET32a-P vector containing staphylococcal protein A gene

[0024] The monomer 2 obtained in the above example 1 was digested with AccI, and the corresponding fragment was recovered, and then connected to the pET32a vector constructed in the above example 2 containing a staphylococcal protein A gene monomer, transformed into Escherichia coli, and screened for the presence of ampicillin. The penicillin-resistant transformant was extracted from the plasmid and screened by enzyme digestion to obtain a staphylococcal protein A vector containing 3 protein A gene monomers, labeled as "pET32a-P". After detection, its gene sequence is shown in SEQ ID No: 2 Show.

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Abstract

The invention discloses novel ProteinA, and a preparation method and use of the novel ProteinA. The ProteinA provided by the invention has the amino acid sequence shown in SEQ ID NO: 1. An affinity chromatography medium prepared from the staphylococcal protein A provided by the invention has the advantages of high protein-binding specificity, high affinity and greatly-increased purification efficiency, so that the separation and purification of protein are better facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Protein A and its preparation method and application. Background technique [0002] Protein A, also known as Staphylococcal Protein A (Staphylococal Protein A, SPA) is a protein isolated from the cell wall of Staphylococcus aureus. 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 A antigen; in 1960, Grov named it staphylococcal protein A, referred to as SPA or 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 is often used as an affinity filler for protein purification because it can specifically bind to the Fc region of various immunoglobulins and can bind to agarose gel in a certain way. With the rapid d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70C12N1/21G01N33/68C07K16/00C07K1/22B01D15/38C12R1/445
Inventor 黎健荣张彦路玲玉胡湘丽
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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