[ presence of insulin catalytic antibodies in plasma of two type 2 diabetes patients]

Recent studies have reported the role of catalytic antibodies in the pathogenesis pattern of some diseases. Autoimmune reactions playing a role in some type 2 diabetic patients and the p- cell autoimmune markers were found to be present in some of these patients. The presence of such antibodies was assessed in the plasma of patients suffering from type 2diabetes. Antibodies in 3 diabetic patients and 7 non-diabetic control subjects were purified using protein-G sepharose affinity column chromatography and S-300 gel filtration methods; purity of the IgG antibodies was confirmed by SDS-PAGE and in western blot analysis and proteolytic activity of electrophoretically homogenous IgG antibodies was confirmed with zymogram analysis. The purified antibodies were incubated with insulin at 37°C for 6 days and the effect of antibodies on insulin degradation was assessed by Acetic Acid-Urea polyacrylamide gel electrophoresis. Insulin degradation effect was observed only in purified antibodies in the 2 diabetic patients and it was not seen in the 7 control subjects and the remaining diabetic patients. Our data revealed, for the first time, insulin degradation by isolated IgG from 2 diabetic patients. This finding may not only explain the insulin resistance observed in some diabetic patients, but may most likely propose also a new mechanism for occurrence of the disease

Studies on the optimal expression condition, purification and its characterization of ScFv-2F3 / 生物工程学报

The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.