Gene engineering method for preparing a recombinant glutathion peroxidase

Abstract

The invention provides a gene engineering method for preparing a recombinant glutathion peroxidase, which belongs to the field of biological technology. The method comprises the following steps: assembling a target gene to a secretion type prokaryotic expression vector, introducing a catalytic group SeCys of GPX into a substrate combination position of GPX by a gene mutant method in an auxotroph prokaryotic expression system or an auxotroph and SPP combined expression system, thereby the activity of the GPS is very high; or assembling the target gene with the SeCys insertion sequence into a secretion type mammalia cell expression vector, assembling a SeCys insertion sequence binding protein 2 into an intracellular type mammalia cell expression vector, and cotransfecting the two vectors into the same lactation cell strain, and synthesizing the GPX under the existence of sodium selenite. The invention the advantages of simple method, recombined GPX vitality, high output and good stability, thereby avoiding decreased yield and inactivation caused by renaturation of an inclusion body, and solving the problem that the natural GPX source is limited and the property is not stable.

Description

technical field [0001] The invention relates to a method for preparing recombinant glutathione peroxidase by using a genetic engineering method, belongs to the field of biotechnology, and relates to gene recombination technology, gene mutation technology and recombinant protein expression technology. Background technique [0002] The substrate of selenium-containing glutathione peroxidase (GPX) is glutathione (GSH), and the catalytic group is selenocysteine ​​(SeCys). In organisms, GPX together with superoxide dismutase (SOD) and catalase (CAT) constitute the body's antioxidant defense system. GPX plays an important role in this system. It uses the substrate GSH as a reducing agent to decompose hydrogen peroxide and various hydroperoxides in the body, so it can remove reactive oxygen species (ROS) in the body and prevent lipid peroxidation. Treat various diseases caused by active oxygen, such as aging, ultraviolet radiation, cardiovascular and cerebrovascular diseases, cata...

AI Technical Summary

Problems solved by technology

[0004] Chinese patents 94102481.4, 96112628.0, 99104234.4, and 200810050556.6 disclose various selenium-containing abzymes. The preparation method is exactly to apply the chemical mutation (modification) method to introduce the catalytic group SeCys of GPX in the antibody template protein, which has the following disadvantages: (1) During the preparation process, only the step of chemical mutation (modification) will lose 20-40% of the enzyme protein, resulting in a significant drop in the yield of the simulated enzyme; (2) The cycle of preparing the simulated enzyme is long, the operation is cumbersome, and the time is long; (3) In the process of chemical mutation, phenylmethylsulfonyl fluoride, acetonitrile, etc. need to be used, these substances are toxic substances; (4) lack of targeting, for large protein molecules, a chemical reaction is often introduced at different sites Multiple non-specific catalytic groups, so the introduction of catalytic groups by chemical mutation cannot achieve the specificity of gene mutation method

[0005] Chinese patent 200810050556.6 also discloses that another preparation method of human single-chain selenium-containing abzyme is to introduce a catalytic group using a defective prokaryotic expression system (Escherichia coli) and gene mutation, but it is limited to human single-chain selenium-containing abzyme Preparation of abzymes, excluding recombinant glutathione peroxidase (GPX) and other GPX mimic enzymes

[0006] Since the codon UGA of SeCys, the catalytic group of GPX, is a stop codon, in the common prokaryotic expression system, it is necessary to introduce a neck loop structure in the open reading frame of the GPX gene, immediately downstream of the codon UGA of SeCys, to translate UGA into SeCys instead of a stop codon, and the introduction of a neck loop in the open reading frame will inevitably cause a change in the spatial conformation of GPX, thereby affecting the enzyme activity

Therefore, the common prokaryotic expression system is not suitable for direct expression of selenoproteins with GPX activity