Hybridized tRNA (transfer ribonucleic acid) and application thereof to glutathione peroxidase preparation

Abstract

The invention discloses hybridized tRNA (transfer ribonucleic acid) and application thereof to glutathione peroxidase preparation and belongs to the field of biotechnology. The hybridized tRNA relates to sequences SEQ ID No: 1-16, consists of 90 basic groups, and not only can serve as substrate tRNA for selenocysteine synthesis, but also can be recognized by autologous elongation factor EF-Tu of escherichia coli. A hybridized tRNA gene is obtained by a gene synthesis method, then a GPX (glutathione peroxidase) gene is synthetized or amplified, the hybridized tRNA gene and the GPX gene are assembled on a secretory prokaryotic expression vector capable of expressing the hybridized tRNA, a TAG engineering strain is transformed and subjected to induced expression in presence of sodium selenite, and a catalytic group Sec of the GPX is introduced into a substrate binding site of protein in a way that conventional amino acids enter a peptide chain, so that the protein is given with high GPX activity. The hybridized tRNA and application thereof to glutathione peroxidase preparation have the advantages that the method is simple, and zymoprotein is high in activity, yield and stability, so that the problems that natural GPX is limited in source and instable in property are solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybrid tRNA and its application in preparing high-activity selenium-containing glutathione peroxidase. Background technique [0002] The substrate of selenium-containing glutathione peroxidase (GPX) is glutathione (GSH), and the catalytic group is selenocysteine ​​(Sec). 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, diabetes, tumors, etc. Different from other antioxidant enzymes, GPX can not only scavenge ROS, b...

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Problems solved by technology

[0004] Chinese patent 94102481.4, 96112628.0, 99104234.4, 200810050556.6 discloses the preparation method of all kinds of selenium-containing abzymes is exactly to apply the chemical mutation (modification) method to introduce the catalytic group Sec of GPX in the antibody class template protein, has following shortcoming: (1) in In the preparation process, only the step of chemical mutation (modification) will lose 20-40% of the enzyme protein, resulting in a significant decline 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 another preparation method of human single-chain selenium-containing abzyme is to use auxotrophic prokaryotic expression system (Escherichia coli) and gene mutation method to introduce catalytic groups, but it is limited to human single-chain selenium-containing Preparation of selenium abzymes, excluding glutathione peroxidase (GPX) mutants and other GPX mimetic enzymes

[0007] The methods disclosed in Chinese patents 201310302778.3 and 2013101428661 have made breakthroughs, but they can only be used to prepare various types of GPX mutants with high activity, and cannot be used to prepare natural GPX proteins

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