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A performance-improved recombinant Staphylococcus aureus protein a affinity ligand and its construction method

A staphylococcal protein and recombinant protein technology, applied in the field of genetic engineering, can solve the problems of difference in binding ability, different elution conditions, and inability to use strong alkali for a long time, and achieve the effect of good binding ability

Active Publication Date: 2015-09-30
嘉兴千纯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although natural protein A has good tolerance to alkaline environment, it still cannot meet the long-term use of strong alkali to clean it.
Although the five domains E, D, A, B, and C of the natural protein A antibody binding region have high homology with each other, their binding ability to IgG is different, resulting in different optimal elution conditions

Method used

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  • A performance-improved recombinant Staphylococcus aureus protein a affinity ligand and its construction method
  • A performance-improved recombinant Staphylococcus aureus protein a affinity ligand and its construction method
  • A performance-improved recombinant Staphylococcus aureus protein a affinity ligand and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The B segment of protein A was modified by overlapping PCR technology, and the modified B segment was named Z segment, and its expressed gene was z. According to the gene sequence provided by GeneBank, use the molecular biology software DNAMAN to design the forward primer F of z sequence 0 and reverse primer R 0 :

[0026] f 0 : CGGATAACAAATTCAAC

[0027] R 0 : TTA GCAACA TTTTGGTGCTTGTGC.

[0028] The forward primer and the reverse primer introduced Noc I and BamH I restriction sites, respectively, where the italic part is the restriction site, and the underlined part is the codon of the two cysteines added.

[0029] Add 6 glycines behind the second loop of the B fragment to reduce the binding force between protein A and IgG, expressed as Z(6G); in order to increase the alkali resistance of protein A, the 23rd asparagine and the 30th asparagine The phenylalanine at the position is replaced by threonine and alanine respectively, represented by Z(N23T, F30A)...

Embodiment 2

[0037] Extract Staphylococcus aureus DNA, utilize the F of embodiment 1 design 0 , R 0 As a primer, Staphylococcus aureus DNA as a template, using Pfu DNA polymerase to perform PCR on the B sequence. According to the instructions of Pfu DNA polymerase, select 50 μL to carry out.

[0038] PCR reaction conditions: pre-denaturation at 94°C for 5min; denaturation at 94°C for 40s, annealing temperature at 56°C for 1min30s, extension temperature at 72°C for 30s, and 30 cycles; finally, hold at 72°C for 10min.

[0039] Fragment B was recovered using an agarose gel recovery kit. Using the method of overlapping PCR, using Pfu DNA high-fidelity polymerase, using the recovered B fragment as a template, respectively using the F fragments designed in Example 1 0 and R 1 , F 1 and R 0 Perform PCR for the primers, and the reaction system and conditions are the same as above. The recovered products of PCR were mixed according to the volume ratio of 1:1, and the mixture was used as a te...

Embodiment 3

[0042] z n Indicates that n (n is 1-10) z sequences are connected end to end. Nhe I and BamHI were used as the first group of restriction endonucleases, and Xba I and BamH I were used as the second group of restriction endonucleases. Overnight culture of recombinant bacteria E.coli JM109 / pMD18-T-z obtained in Example 2 1 , plasmid extraction pMD18-T-z 1 , using two sets of enzymes to recombine the recombinant plasmid pMD18-T-z 1 Carry out enzyme cleavage reaction. The two groups of digested products were recovered from the gel, and the two groups of products were ligated overnight at 16°C using T4 DNA ligase to obtain a recombinant plasmid pMD18-T-z containing two z sequences connected end to end 2 , the pMD18-T-z 2 Transformed into competent E.coli JM109 for preservation.

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Abstract

The present invention discloses a performance improved recombination staphylococcus aureus protein A affinity ligand and a construction method thereof. The present invention adopts a molecular biology method. A sequence B of a nature protein A is selected for molecular transformation. A C-terminal of the sequence B is added with two cysteines, so that the protein A can pass through double-locus coupled chromatography matrix to stabilize the connection. Six glycines are added to the end of a second Loop of the sequence B to increase the length and reduce the binding force with an antibody, so that elution conditions are mild. On this basis, resistance performance to high concentration base of the protein A is transformed. Asparagines and phenylalanine at 23rd and 30th positions of the sequence B are respectively replaced by threonine and alanine to obtain a sequence Z with higher alkaline resistance properties. Then, isocaudarner is used for connecting sequence Zs of different numbers head-to-tail in series. The efficient expression system of e. coli is used for overexpression. The expressed recombination protein A is coupled to agarose matrix preparation affinity chromatography fillers and is used for purifying antibodies. Results show that the recombination protein A affinity ligand prepared by the present invention is good in elution performance and alkali resistance.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a performance-improved recombinant Staphylococcus aureus protein A affinity ligand and a construction method thereof. Background technique [0002] Staphylococcus aureus protein A, referred to as protein A, is a protein on the surface of Staphylococcus aureus, which is covalently linked to the cell wall peptidoglycan. There are five highly homologous E, D, A, B, and C sequences in the protein A molecule, which are called antibody binding regions because they can bind to the Fc region of human and various mammalian IgGs. The binding of protein A to the Fc region of the antibody does not affect the adsorption activity of the antibody to the antigen, so it is widely used in the affinity purification of antibodies. Since the B domain has the most stable binding force to antibodies and its structure is also the most stable, people have studied the B sequence the most. The B sequence ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/31C12N15/31C12N15/70C12N15/10C12R1/445
Inventor 夏海锋吴璞强梁振东王沙莉郑梦杰
Owner 嘉兴千纯生物科技有限公司
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