A new autoantigen reactive with prediabetic nonobese diabetic mice sera

The identification and characterization of new autoantigens would widen the knowledge of the pathogenic mechanism of insulin dependent diabetes mellitus. Screening of lambda gt11 mouse insulinoma (MIN6N8a) cell cDNA library with prediabetic nonobese diabetic (NOD) mice sera resulted in the isolation of a strong positive clone, named the clone 3-5, of 1579 nucleotides without a poly A region. After 5'-rapid amplification of the cDNA end (RACE), complete nucleotide sequence of the clone 3-5 gene consisting of 2231 nucleotides showed that the 3-5 gene had the theoretical open reading frame of 634 amino acids. However, the real antigenic protein of the clone 3-5 was only 21 amino acids long encoded by only 63 nucleotides. The 21 amino acids were expressed as a fusion protein in E. coli and purified by affinity chromatography. The purified 3-5 recombinant protein was examined for its reactivity with prediabetic NOD mice sera by immunoblotting. The only non-denatured form of the 3-5 protein showed a binding reactivity with NOD mice sera, demonstrating that the conformational epitope of 3-5 protein was important for antibody recognition. The prevalence of autoantibody reactive to the 3-5 protein was about 78% (14/18) and 46% (11/24) in prediabetic and acute diabetic NOD mice sera, respectively. However, the sera from other mouse strains such as BALB/c, ICR, C57BL/6, SJL/J, and NOD/SCID did not show a positive reactivity to the 3-5 protein, which indicated that immune reactivity against the 3-5 protein was autoimmune diabetic mouse-specific.