Verfahren zur Herstellung hochreiner humaner GAD-1- und GAD-2- Proteine

摘要

The invention pertains to a process for producing high-purity human GAD-1 and GAD-2 proteins using the baculovirus/Sf9 expression system; the process comprises the following steps: a) preparation of cDNA sequences from human cDNA libraries which code GAD-1 and GAD-2 proteins at full length or in modifications thereof; b) insertion of the GAD-1 or GAD-2 cDNA sequence into a baculovirus transfer vector; c) co-transfection of Sf9 cells with baculovirus transfer vectors containing GAD-1 or GAD-2 cDNA and with baculovirus DNA to produce recombinant baculoviruses; d) identification, selection and enrichment of recombinant baculoviruses; e) extraction of GAD-1 or GAD-2 protein after infection of Sf9 cells with recombinant baculoviruses, and f) purification of the recombinant GAD-1 or GAD-2 protein. The process as per the invention provides high-purity, nearly homogeneous GAD-1 or GAD-2 proteins with a high rate of expression and a high degree of purity. The isolated GAD proteins are used in immunoassays for early detection of type I diabetes mellitus (IDDM).