| Oxidative Stress is a kind of unbalance state between oxidation and antioxidation. Reactive oxidative species (ROS) are harmful to cells when oxidative metabolism products increase or the antioxidative defenses lack in the organism. Lots of researches showed that ROS are related to some physiologic and pathological processes and act important effects in many diseases. ROS have highly activity and very strong oxidative ability and they can attack the molecules by the oxidation, and then make the big molecules denaturation, cross-linking and breakout, and then lead to the damage of cell structure and function and the injury of the tissue. There are two kinds of the scavengers for free radicals in the organism, namely enzymes (Antioxidative enzymes) and non-enzyme compounds. The enzyme scavengers main include SOD, CAT and GPX, and they form the defense system in the organism against free radicals. In addition, many small molecular compounds also have strong antioxidative ability. These compounds are vitamin C, vitamin A, A-tocopherol, ascorbic acid, reduced glutathione, β-carotene, metal chelate complex, calcium ion antagonists, flavonoid, polyphenol compouns and antioxidant peptides, and so on. They act with antioxidant enzymes to make free radicals under a normal level of metabolism in the organism. GPX is a kind of protein containing selenium. Although the structure of GPX and the biologic effect of selenium have been understood fully, the effect mechanism of GPX needs to be studied further. The GPX mimics can be used to do this. On the other hand, GPX has strong antioxidant ability and it is important for treat and prevent Keshan disease, angiocardiopathy, inflammation and cancer. Due to the limitations associated with native GPX, such as instability, limited availability, big molecular weight and immunogenicity, many scientists have made a great deal of efforts to study the GPX mimics.Due to the lack of the GSH binding site, the early GPX mimics have generally rather low activities. Take ebselen for example, its activity only has 0.99U/(mol. Wilson et al. introduced quaternary ammonium salt near to diselenide bridge of GPX mimic to improve the ability to bind GSH by electrostatic attraction to GSH and quaternary ammonium salt can provide proton for reduction of another substrate, H2O2. So the activity of this kind of GPX mimic, dipheyl diselenide compound, can be increased up to 11U/(mol. Hilvert et al. turned subtilisin to GPX mimic by chemical mutation. The catalytic efficiency of the mimic was 7(104 times more than that of dipheyl diselenide compounds using the favored 3-oxatyl-4-nitryl phenthiol as substrate. But its activity towards GSH, the substrate of native GPX, was rather low. So the key of imitation GPX is to generate a specific binding site for GSH. According to the idea, we have developed several selenium-containing catalyzed antibodies. They had high catalytic activities, but there are some drawbacks for use as drugs, such as the big molecular size led to the difficulty for transport in organism and penetration into cells. So we decided to construct a small molecular GPX mimic with high activity base on the specific combination with substrate and research its enzymologic character and biologic efficacy. We confirmed the biologic efficacy of the GPX mimic in organism through the protective effect of the GPX mimics in the cells. 1．Construction of selenium-containing single-chain Fv to imitate GPX We had prepared successfully several selenium-containing antibody enzymes by the technology of preparation monoclonal antibody. But due to the big molecular weight, selenium-containing antibody is not suitable for pharmaceutical development. So we constructed the vector with T7RNA polyase strong promotor, pTMF-scFv, to express single-chain Fv fragment (scFv) as an inclusion body form. Due to the difficulty of renaturating the inclusion body, and the rather low rate of production, the preparation of selenium-containing single-chain Fv is rather difficult. In order to resolve t...
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