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Preparation of recombinant protein a gene and its expression product

A recombinant protein and gene expression technology, applied in the field of bioengineering, can solve the problems of little influence on the activity of protein A ligand, great harm to the human body and the environment, and low activation efficiency, and achieve strong binding specificity, short expression time, and high purity and high yield

Active Publication Date: 2015-12-09
AGTC GENE TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The currently used protein A immunoadsorption material uses agarose gel as the matrix, and the coupling method with protein A is mostly activated coupling with cyanogen bromide or epichlorohydrin. These two activation methods have obvious shortcomings: (1 ) cyanogen bromide is a highly toxic substance, and the synthesis process is harmful to the human body and the environment; (2) when epoxy is activated, the epoxy group is easily hydrolyzed under alkaline conditions, and the activation efficiency is low; this study uses N-hydroxy The succinimide active esterification method (NHS) activates the carrier, the activation conditions are mild, and has little effect on the activity of the protein A ligand, and because the spacer arm is introduced during the activation process, the protein A affinity filler per unit volume can bind more IgG, can significantly increase its IgG binding capacity

Method used

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  • Preparation of recombinant protein a gene and its expression product
  • Preparation of recombinant protein a gene and its expression product
  • Preparation of recombinant protein a gene and its expression product

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Synthesis of embodiment 1 recombinant protein A gene monomer

[0031] In order to promote the expression of the Staphylococcus aureus protein A gene in Escherichia coli, according to the codon preference of Escherichia coli, some base sequences in the B domain gene of protein A were optimized (there are multiple optimization schemes, sequence table SEQNo. 1 is one of the optimized sequences), through the method of chemical synthesis, the recombinant protein A gene monomer sequence was designed and synthesized. Connectors for body connection. In addition, it also includes start codon ATG, stop codon TAA, EcoRI and BamHI restriction endonuclease sites for cloning, and 6×His tag sequence at the 5′ end. Embodiment 2 Contains the construction of the pBV220 expression vector of a recombinant protein A gene monomer

Embodiment 2

[0032] The chemically synthesized recombinant protein A gene monomer was digested with EcoRI and BamHI and connected to the pBV220 vector with the same restriction enzymes, transformed into Escherichia coli, and the transformant with ampicillin resistance was screened, and the plasmid was extracted by PCR and digestion And after sequencing and identification, it was proved that the recombinant protein A gene monomer had been cloned into pBV220.

Embodiment 3

[0033] Embodiment 3 Contains the construction of the pBV220-P expression vector of recombinant protein A gene

[0034] The above-mentioned pBV220 vector containing a recombinant protein A gene monomer and the recombinant protein A gene monomer were digested with AccI, and the corresponding fragments were recovered and ligated, transformed into Escherichia coli, and the transformant with ampicillin resistance was screened, and the plasmid was extracted by After identification by PCR, enzyme digestion and sequencing, it was proved that the recombinant protein A gene (six protein A gene monomers connected in series) had been cloned into pBV220. The result is attached to the manual figure 1 shown.

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Abstract

The invention relates to the technical field of biological engineering. The B structure domain gene monomer of protein A is optimized according to the preference of a colon bacillus colon, six series bodies are constructed and inserted into a thermally induced colon bacillus vector pBV220, and a colon bacillus expression vector containing the gene and a converted colon bacillus DH5alpha recombination strain thereof are constructed. And a method for producing the recombination protein A using the strain is also provided. The total soluble bacterial protein amount of the soluble recombination protein A produced by the strain can be more than 80 percent, and the recombination protein A can be purified to over 95% just by subsequent nickel ion chelate chromatography and molecular sieve column chromatography. Moreover, the protein has the advantages of high expression amount, low cost, easy purification and the like. A recombination protein A affinity stuffing prepared by using the protein has the advantages of high human IgG absorption capacity, low proA dropping and the like. The invention provides a practical and feasible approach to obtain the recombination protein A with high quality and low price as an antibody (medicine) purifying medium.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and relates to a recombinant protein A gene, a vector containing the recombinant gene, a transformed bacterial strain and a product expressed by the recombinant protein A gene, a purification method thereof, and preparation and use of an affinity filler. Background technique: [0002] Staphylococcal Protein A (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 also discovered a similar phenomenon in 1959 and named it the A antigen. In 1963, Lofkvist and others isolated substance A, proving that it is a protein and different from sugar; in 1960, Grov named it staphylococcal protein A, referred to as SPA (ProteinA). The gene encoding SPA was cloned in 1983 and expressed in E. col...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/70C07K14/31C07K1/22
Inventor 宫照龙何新舟曹晖许允立
Owner AGTC GENE TECH CO LTD
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