Preparation of virus-like particle mimetic nanovesicles displaying the S protein of Middle East respiratory syndrome coronavirus using insect cells

In this study, structural proteins of MERS-CoV were expressed in silkworm larvae and Bm5 cells for the development of vaccine candidates against MERS-CoV and diagnostic methods. The spike (S) protein of MERS-CoV lacking its transmembrane and cytoplasmic domains (SοTM) was secreted into the hemolymph of silkworm larvae using a bombyxin signal peptide and purified using affinity chromatography. The purified SοTM forms small nanoparticles as well as the full-length S protein and has the ability to bind human dipeptidyl peptidase 4 (DPP4), which is a receptor of MERS-CoV. These results indicate that bioactive SοTM was expressed in silkworm larvae. To produce MERS-CoV-like particles (MERS-CoV-LPs), the coexpression of spike proteins was performed in Bm5 cells and envelope (E) and membrane (M) proteins secreted E and M proteins extracellularly, suggesting that MERS-CoV-LPs may be formed. However, this S protein was not displayed on virus-like particles (VLPs) even though E and M proteins were secreted into the culture supernatant. By surfactant treatment and mechanical extrusion using S protein- or three structural protein-expressing Bm5 cells, S protein-displaying nanovesicles with diameters of approximately 100⿿nm⿿⿿⿿200⿿nm were prepared and confirmed by immuno-TEM. The mechanical extrusion method is favorable for obtaining uniform recombinant protein-displaying nanovesicles from cultured cells. The purified SοTM from silkworm larvae and S protein-displaying nanovesi...
Source: Journal of Biotechnology - Category: Biotechnology Source Type: research