Journal: JCI Insight

Article Title: A T cell–based SARS-CoV-2 spike protein vaccine provides protection without antibodies

doi: 10.1172/jci.insight.155789

Figure Lengend Snippet: ( A ) Schematic diagram of plasmid design. The gene sequence encoding the S protein was split into 3 parts (denoted S-a, S-b, and S-c). The 30 nucleotide bases before and after any cleaved site were placed back to preserve any epitopes that may have been disrupted. The open-reading frame gene encoding a human monomer of ubiquitin (Ub) was placed immediately upstream of the rearranged S sequence. The gene encoding a glycine at the 76th residue was modified to encode an alanine to enhance the stability of the Ub-S complex. ( B ) The plasmids were digested with EcoRI and NotI and run in an agarose gel. ( C ) 293T cells were transfected with the Ub-S plasmid encoding ubiquitinated and rearranged S protein, a Ub-S Unmodified plasmid encoding ubiquitinated original S protein, or a plasmid encoding original S protein (no ubiquitin), overnight. The cells were allowed to stably express plasmid for 36 hours. After this period, MG132 was added overnight (right). The cell lysate was analyzed via Western blotting for the expression of SARS-CoV-2 S protein and β-actin as a control. Lanes were run on the same respective gels, but are noncontiguous.

Article Snippet: This nucleotide sequence was ordered from GenScript.

Techniques: Plasmid Preparation, Sequencing, Ubiquitin Proteomics, Residue, Modification, Agarose Gel Electrophoresis, Transfection, Stable Transfection, Western Blot, Expressing, Control