Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells / 生物工程学报

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Oxidative stress and vascular endothelial growth factor in rats with type 1 diabetes mellitus / 医学研究生学报

O bjective Different degrees of oxidative stress may exist in patients with diabetes mellitus (DM).The aim of this study was to explore the roles of oxidative stress and the level of vascular endothelial growth factor ( VEGF) in the development and progression of DM in the rat model of type 1 DM (T1DM). Methods We established the T1DM model in 23 male SD rats and in-cluded another 17 in the control group .We determined the total anti-oxidation capacity ( T-AOC) and the levels of super-oxide dis-mutase (SOD), glutathione (GSH), and malondialdehyde (MDA) by chromatometry, and measured that of VEGF by ELISA. Results At 7 weeks after modeling, the level of fasting blood glucose(FBG) was significantly increased in the T1DM rats as com-pared with the controls ([23.01 ±3.62] vs [5.08 ±0.46] mmol/L, P0.05).FBG was correlated positively with MDA (r=0.51) and VEGF (r=0.47) but negatively with GSH (r=-0.71) and SOD (r=-0.40), while serum VEGF positively with MDA (r=0.32) and negatively with GSH ( r=-0.34 ) in the experimental rats . Conclusion Peroxidation is increased and the anti-oxidation mecha-nism weakened during the development and progression of T 1DM, which is closely related to hyperglycemia .High VEGF is associated with hyperglycemia and enhanced peroxidation .