2.1 Recombinant virus construction
The design of the chimeric molecule contained the coding sequence of the C-terminal domain of human ZnT8, which consisted of a dimer that includes amino acids 268–369 [R325] and amino acids 268–369 [W325], together with full-length human GAD65. Also, a histidine-hexapeptide (His6) tail at the N-terminus was added. This coding sequence was synthesized by GenScript Corporation (Piscataway, NJ, USA; www.GenScript.com). Codon optimization for insect cell expression was done.
The chimera cassette was directly cloned under the polyhedrin (polh) promoter using the EcoR1 and XbaI site, into the pFastBac™ Dual vector (Thermo Fisher Scientific), previously modified with the enhanced green fluorescent protein (EGFP) cDNA under the p10 promoter (Targovnik et al., 2019). The final construction pFBD‐p10‐EGFP‐polh‐ZnT8/GAD65 is shown in Figure 1.
The recombinant baculoviruses were obtained using the Bac to Bac™ baculovirus expression system (Thermo Fisher Scientific), following the manufacturer’s instructions, as we have previously described (Smith et al., 2022). Minor modifications were done in order to generate the desired virus. Once the bacmids were purified from Escherichia coli DH10Bac™ strain (Thermo Fisher Scientific), and before transfecting Sf9 cells, the sequences were verified by Sanger sequencing (GenBiotech). The transfection was evaluated by the green fluorescence generated under UV light (Figure 2). After 4 days of infection, the culture supernatant was recovered. The recombinant virus was named Acpolh‐ZnT8/GAD65.
Finally, amplification was performed at a multiplicity of infection (MOI) of 0.02 in Sf9 cells seeded in T‐25 flasks at 27°C. The amplified Acpolh‐ZnT8/GAD65 was titrated by plaque assay (O’Reilly et al., 1994). This high‐titer Acpolh‐ZnT8/GAD65 was the viral stock used for protein production in insect cell.
The Sf900 II insect cell culture medium was from InvitrogenTM (Gaithersburg) and the antibiotic and antimycotic solutions (Anti-Anti 100X) and the fetal bovine serum were from GIBCO (Thermo Fisher Scientific).