3.1 Generation of the Recombinant Baculovirus Acpolh-ZnT8/GAD65
For the construction of the chimera, we based ourselves in the knowledge
of the immunodominant regions of both proteins. As previously described
by Rickert et al. (2001), GAD65 has nine conformational epitopes
throughout its entire structure so we decided to use full length GAD65.
As for ZnT8, it is known that C-terminal domain (amino acids 268–369)
is the region most recognized by specific autoantibodies (Wenzlau et
al., 2007). Also, this region contains a polymorphism in amino acid 325.
In this sense, we decided to use a heterodimeric variant of C-terminal
domain containing R or W in 325 position, based on that we have
previously demonstrated that this heterodimeric construction showed
higher signal levels and increased dynamic range in RBA for ZnT8A
detection (Faccinetti et al., 2016).
The cassette containing a dimer of ZnT8 and full length GAD65, fused to
a His-tag was cloned under the control of the polyhedrin promoter, hence
pFBD-polh-ZnT8/GAD65 was obtained. The N-terminal His-tag was added to
facilitate the purification of the recombinant chimera by
Ni2+–NTA Immobilize Metal Affinity Chromatography.
The expression vector used in this work has another distinctive feature,
which is the employment of EGFP, cloned under the p10 promoter, as a
reporter gene useful for visualizing viral infection. The corresponding
bacmids were produced with these plasmids. The Acpolh-ZnT8/GAD65 virus
was finally obtained by transfection of the bacmids and amplification in
Sf9 cells.