Expression based on baculovirus system of properly folded autoantigens -GAD65 and IA-2ic

TRABUCCHI A, ROVITTO BD, FACCINETTI NI, TARGOVNIK AM, GUERRA LL, IACONO RF, POSKUS E, MIRANDA MV, AND VALDEZ SN

Buenos Aires

Congreso; IV LASID Meeting, LXIII Argentinean Society for Immunology Meeting and II French Society for Immunology Meeting; 2015

Argentinean Society for Immunology and French Society for Immunology

Expression based on baculovirus system of properly folded autoantigens -GAD65 and IA-2ic The aim of this work was to express two relevant autoantigens useful in the diagnosis of Autoimmune Diabetes Mellitus, glutamic decarboxylase (GAD65) and the intracellular fraction of IA2 (IA2ic), in a eucariotic system based on baculovirus.Methods: GAD65 and IA2ic were expressed using the baculovirus expression system based on SF9 insect cells and on Rachiplusia nu larvae. SF9 cells were infected at a multiplicity of infection of 0.5 and 1 pfu/cell for GAD65 and IA2ic expression, respectively. Fourth-instar R.nu larvae were infected with 1Ã?107 pfu/ml and homogenized with extraction buffer. The expression of proteins was assessed by Western blot using specific antibodies. Immunoreactivity was evaluated by radioimmunoassay using [35S]GAD or [35S]IA2 as tracers and recombinant GAD65 or IA2ic as competitors, in presence of diabetic patients sera. Results: A major band of 66kDa and another of 43KDa were detected both in pellets of SF9 cells and larval extracts, in agreement with the molecular weight expected for GAD65 and IA2ic, respectively. Antigens expressed in SF9 cells were recovered and purified from intracellular soluble fraction, while they were recovered from the total homogenate of infected larvae. The radioimmunoassay showed a decrease in the signal from 8.00% to 4.27% with SF9-GAD65, from 11.15% to 1.92% with SF9-IA2ic, from 7.70% to 1.76% with larvae-GAD65 and from 8.40% to 5.30% with larvae-IA2ic.Conclusions: The recombinant autoantigens GAD65 and IA2ic were properly expressed in both, SF9 cells and R.nu larvae, holding their immunochemical structure associated to conformational epitopes that are recognized by diabetic patients sera.