Molecular cloning and prokaryotic expression of IA-2 gene

Objective To get sufficient IA-2 recombinant protein with immunoreactivity for the assay of IA-2 autoantibodies. Methods The gene encoding the intracellular domain of IA-2 was amplified from neonatal mouse brain tissue RNA by RT-PCR, and was subcloned into Pinpoint Xa-1 T vector to construct recombinant expression plasmid. After sequence and open reading frame confirmed by DNA sequencing, the expression of target DNA in E. coli was induced. By using avidin resin, the biotinylated fusion protein (molecular weight about 56 000) was affinity-purified under non-denaturing conditions. Thereafter, its immunogenicity was identified by dot blot using the sera of positive glutamic acid decarboxylase antibody from 10 diabetic patients as compared with the sera from healthy controls. Results The PCR products were correctly inserted into restriction sites of expression vector. The overall yield of purified recombinant protein was about 1 mg/L of bacteria culture. The reaction of IA-2 recombinant protein with GAD antibodies positive sera of diabetic patients was significantly different from those with sera of healthy controls. Conclusion The expressed IA-2 fusion protein shows expected immunoreactivity and will be used in further researches on type 1 diabetes.