Recombination protein A gene and preparation of expression product thereof

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...

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

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