Preparation of soluble human selenium-containing single-chain abzyme

Abstract

The invention discloses a method to prepare soluble human selenium-containing catalytic single-chain antibodies, belonging to biotechnology field. The method includes the following steps: with glutathione derivatives as target antigen, filtrating the recombinant phage display human single-chain antibody library through immune affinity selection method to obtain single-chain antibodies B3 and D8; assembling the single-chain antibodies to secretory procaryon or eucaryon expression vector; translating colon bacillus or yeast cell; expressing and purifying the single-chain antibody proteins in procaryon or eucaryon; introducing GPX catalytic group SeCys at the substrate combining sites of the antibodies through chemical mutation method or directly expressing the soluble single-chain antibody proteins which contain GPX catalytic groups at the substrate combining sites with auxotrophic colon bacillus through genetic mutation techniques to endue the antibodies with GPX catalytic activity. The method of the invention is simple and the human selenium-containing catalytic single-chain antibodies prepared with the method are of high catalytic activity; the proteins expressed by the antibodies are soluble; therefore the method is good for large-scale production and is of broad application prospect in biological pharmacy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing glutathione human source selenium-containing single-chain antibody enzyme by applying the technology of combining genetic engineering and chemical mutation. 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 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. Various diseases caused by active oxygen, such as ultraviolet radiation, cardiovascular and cerebrovascular diseases, cataracts, tumors, etc. Different from other an...

AI Technical Summary

Problems solved by technology

The method for the human source selenium-containing single-chain antibody enzyme prepared by this method has the following disadvantages: (1) the inclusion body protein expressed in the preparation process is a kind of inactive protein molecule without normal spatial conformation and in a precipitated state. Denaturation - renaturation to be active

The renaturation of inclusion body protein is a difficult and complicated process. Only in the renaturation step will a large amount of single-chain antibody protein be lost, resulting in a decrease in the yield of antibody enzymes prepared by this method; (2) the preparation cycle of antibody enzymes is long and the operation is cumbersome , because inclusion body renaturation is a slow process to gradually restore activity of inactive protein molecules under suitable conditions; (3) The activity of the prepared antibody enzyme is low, only 28.8-256U / μmol, which is an order of magnitude lower than the natural enzyme