Method for preparing A gene of recombined protein, and expressed products and application

A recombinant protein and gene technology, applied in the biological field, can solve the problems of low affinity and low purification efficiency, and achieve the effects of high affinity, high expression efficiency and short expression time.

Active Publication Date: 2007-10-10
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

When using the affinity chromatography technology containing proteinA, there are problems such as low affinity and low purification efficiency. Therefore, wh

Method used

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  • Method for preparing A gene of recombined protein, and expressed products and application
  • Method for preparing A gene of recombined protein, and expressed products and application
  • Method for preparing A gene of recombined protein, and expressed products and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Synthesis of Recombinant Protein A Gene Monomer

[0027] By means of chemical synthesis, the sequence of a repetitive fragment of the recombinant protein A gene was designed and synthesized. The sequence included a length of 168 bp, and the NcoI restriction site connected to the expression vector was added to the front end, and 6 His (for affinity chromatography, and Nickel column combination, reducing purification steps) and EK restriction site, and AccI restriction site for ligation of 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. It is also necessary to design and synthesize a sequence of a repeat segment of the recombinant protein A gene by chemical synthesis, which has a length of 168 bp and AccI restriction sites at both ends for constructing a sequence containing multiple repeat segments.

Embodiment 2

[0028] Example 2 Construction of a pET32a vector containing a recombinant protein A gene monomer

[0029] Cut out the above-mentioned recombinant protein A gene monomer with restriction endonucleases NcoI and BamHI, connect with the vector pET32a digested with NcoI and BamHI, transform Escherichia coli, screen the transformant with ampicillin resistance, and extract the plasmid , After identification by enzyme digestion, it was proved that the recombinant protein A gene monomer had been cloned into pET32a.

Embodiment 3

[0030] Example 3 Construction of pET32a-P vector containing recombinant protein A gene

[0031] The above pET32a vector containing a recombinant protein A gene monomer and the recombinant protein A gene monomer were digested with AccI, the corresponding fragments were recovered and ligated, transformed into Escherichia coli, and the transformants with ampicillin resistance were screened, extracted from the plasmid, and enzymatically The pET32a-P vector of recombinant protein A containing 3 protein A gene monomers was obtained by cleavage screening.

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Abstract

This invention provides a recombinant protein A gene, whose nucleotide acid sequence is shown in SEQ ID NO.1, and the recombinant protein A coded by the gene. This invention also provides recombinant expression carrier PET32a-P for expressing the recombinant protein A gene, E. coli BL21/DE3 converted by the gene, and the method for preparing recombinant protein A. Recombinant protein A can be used for antibody detection, separation and purification, and can constitute affinity chromatography medium together with chromatography medium carrier. Recombinant protein A has such advantages as high protein combination specificity, high affinity and high purification efficiency, and has obviously increased dynamic adsorption capacity compared with commercialized protein A sepharose 4 fast flow.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant protein A gene, a carrier containing the recombinant gene, a transformed bacterial strain and a product expressed by the recombinant protein A gene, and a preparation method and application thereof. Background technique [0002] Staphylococcal 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. Jensen (1959) also discovered a similar phenomenon and named it the A antigen. [0003] In 1963, Lofkvist et al. isolated the A antigen, and proved that it is a protein and is different from sugar; Grov (1960) named it staphylococcal protein A, referred to as SPA protein A (ProteinA). The gene encoding SPA was cloned and expressed in Escherichia coli in 1983 (Duggleby, C.J and Jon...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/31C12N15/63C12N1/21C12P21/02G01N33/68C07K16/00B01D15/08
Inventor 谈珉胡辉郭亚军
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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