Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus

Abstract

Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients’ sera in the presence of 1 μM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients’ sera were performed by adding recombinant GAD65 (0.62 nM–1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in insect larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.

Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells

“…Previous studies have shown inefficient secretion of some other recombinant glycoproteins produced by BEVS (Klaus et al 2015;Trabucchi et al 2019). However, the fact that the expressed protein remained inside the cells did not affect its production and recovery.…”

Expression of a ZnT8/GAD65 chimera in insect cells useful for Autoimmune Diabetes diagnosis

“…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.…”

“…After 4 days of incubation in the dark at 27 degC, in continuous agitation, cells were centrifuged. The recombinant chimera was recovered following the protocols of lysis and purification previously described by our group (Trabucchi et al, 2019).…”

“…The protocols followed for SDS-PAGE and WB analysis were the same as Faccinetti et al (2017) and Trabucchi et al (2019). Briefly, protein fractions were separated by 10% SDS-PAGE under reducing conditions, followed by Coomassie Brilliant Blue R-250 staining.…”

“…Biotinylation of the purified chimera was done as we have previously described (Trabucchi et al, 2019, Trabucchi et al, 2022. Two hundred eighty micrograms (280 ug) of desalted protein were incubated with excess of sulfo-NHS-biotin (Pierce Biotechnology), and free biotin was then removed.…”