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Artificial recombinant penton protein A, construction method and use thereof

A technology of recombinant protein and pentaplex, which is applied in the field of genetic engineering, can solve the problems that cannot meet the application requirements of antibody separation and purification, specific sequence differences, and experimental data cannot be seen.

Inactive Publication Date: 2008-11-05
GUANGZHOU KONCEN BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the number of amino acid residues of the two recombinant proteins is relatively close, their specific sequences are significantly different
In the description of the third patent application, you can see the performance comparison between the recombinant protein A and the commercially available natural product; while in the description of the first two patent applications, you can not see the experimental data related to the comparison
Based on the comparative study of the patented products, the inventors found that the performance of these products is actually weaker than that of natural products, so they cannot meet the application requirements of antibody separation and purification, especially with the rapid development of immunoadsorption blood purification technology, the The antibody adsorption performance of protein A puts forward higher requirements

Method used

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  • Artificial recombinant penton protein A, construction method and use thereof
  • Artificial recombinant penton protein A, construction method and use thereof
  • Artificial recombinant penton protein A, construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Acquisition of Recombinant Protein A Gene Monomer

[0032] Design three single-stranded DNA primers, the nucleotide sequences of which are SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 (see the sequence list), which are synthesized by professional companies and spliced ​​by PCR reactions to obtain recombination The protein A gene monomer contains a BamHI restriction site at the 5' end of the monomer and a HindIII restriction site at the 3' end, and then connects the gene monomer into the vector pQE_TriSystemHisStrep1 by restriction enzyme connection to obtain a recombinant protein The recombinant vector pQE-spa-b of A gene monomer.

Embodiment 2

[0033] Embodiment 2: Acquisition of recombinant protein A gene and construction of expression vector containing recombinant protein A gene Design the first pair of primers:

[0034] CL1: 5'-gggCCATGGGTGcggataacaaattcaacaaag-3'

[0035] CL2: 5'-cccGTCGACttttggtgcttgCgcatc-3'

[0036] Design the second pair of primers:

[0037] CL3: 5'-cccGTCGACaacaaattcaacaaagaac-3'

[0038] CL4: 5'-gggCTCGAGttttggtgcttgCgc-3'

[0039] Use these two pairs of primers to amplify gene monomers respectively, the 5' and 3' ends of the amplified product CL1-2 have NcoI and SalI recognition sites respectively, and the 5' and 3' ends of CL3-4 have SalI respectively and XhoI recognition sites. CL1-2 and CL3-4 were ligated after digestion with SalI to obtain a recombinant protein A gene composed of two gene monomers connected in series, which was named spa-b2. spa-b2 was digested with NcoI and XhoI and ligated with the vector pQE_TriSystemHisStrep1 to obtain the recombinant vector pQE-spa-b2 contain...

Embodiment 3

[0041] Example 3: Construction of Escherichia coli recombinant strain pQE-spa-b5-BL21 (DE3) capable of highly expressing recombinant protein A

[0042] The recombinant vector pQE-spa-b5 was transformed into Escherichia coli BL21(DE3), and the recombinant strain pQE-spa-b5-BL21(DE3) with ampicillin resistance was screened according to conventional methods.

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PUM

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Abstract

The invention, pertaining to the field of genetic engineering techniques, relates to an artificially reconstituted penton protein A and a constructing method and usage thereof. The artificially reconstituted penton protein A of the invention has an amino acid sequence shown in SEQ ID NO.1 of the sequence listing. The activity of the reconstituted protein A of the invention exceeds that of the reconstituted protein A reported and reaches the level of natural protein A with high production and relatively simple purification technique, thus avoiding the danger of pathogenic bacteria in naturally extracted proteins and being able to produce in a large scale.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an artificially recombined pentaplex protein A as well as its construction method and application. Background technique [0002] The discovery of protein A was associated with IgG (immunoglobulin G) from the beginning. As early as 1940, Vevwey found that in some Staphylococcus aureus, it contained a protein that could form a precipitate with normal human serum in a two-way diffusion test. substance. Jensen (1959) also found a similar phenomenon and named it A antigen. It was not until 1963 that Lofkvist et al. isolated the antigen and proved that it is a protein. To distinguish it from sugar, Grov (1960) named it staphylococcal protein A, referred to as SPA or protein A. In 1978, some researchers used heat-inactivated and formalin-fixed Staphylococcus aureus for the treatment of a type of cancer, which showed certain curative effect. This was the first time in the wo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/63C12N1/21C12N15/10C12P21/02C07K1/22
Inventor 陈校园余波光彭涛张旭锋
Owner GUANGZHOU KONCEN BIOSCI
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