pCAGGS

Case Study 1

Here, researchers report an electrochemical system for the detection of specific antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in patient serum samples following coronavirus disease 2019 (COVID-19). To this end, the recombinant SARS-CoV-2 spike protein (SCoV2-rS) was covalently immobilised on the surface of the gold electrode pre-modified with mixed self-assembled monolayer (SAMmix) consisting of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. The affinity interaction of SCoV2-rS with specific antibodies against this protein (anti-rS) was detected using two electrochemical methods: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The developed electrochemical immunosensor is suitable for confirming COVID-19 infection or the immune response after human vaccination.

Figure 1. Schematic representation of experimental stages. pCAGGS (Creative Biogene, cat. no. VET1375) is involved in the preparation of the SCoV2-rS protein. (Liustrovaite V, et al., 2022)

The SCoV2-rS protein is produced in mammalian Chinese hamster ovary (CHO) cells as a secreted trimeric protein. To match the native conformation locked in the prefusion state, the gene encoding the extracellular domain of SARS-CoV-2 Spike (SCoV2-S), including amino acids (aa) 1-1208, was chemically synthesized. The synthesized gene was then cloned into the mammalian expression vector pCAGGS (Creative Biogene, catalog number VET1375) by introducing NotI and XhoI restriction sites at the 5' and 3' gene ends, respectively. The entire expression construct includes full-length SCoV2-S ectodomain (aa 1–1208) w/o transmembrane and cytoplasmic aa, furin cleavage site 'RRAR' mutated to 'GSAS', C-terminal GSN4 trimerization motif fused to protein sequence, followed by thrombin cleavage site. Two mutations (K986P and V987P) were also introduced into the SCoV2-S sequence to stabilize the trimer in the prefusion conformation.

Case Study 2

Serological diagnosis and vaccination effectiveness assessment of coronavirus disease 2019 (COVID-19) is determined by the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies. Here, researchers present an electrochemical-based biosensing technology for the detection of SARS-CoV-2 protein-specific antibodies. Recombinant SARS-CoV-2 spike protein (rSpike) was immobilized on the surface of a self-assembled monolayer (SAM)-modified gold electrode. The modified electrode was used as a sensitive element for the detection of polyclonal mouse antibodies against rSpike (anti-rSpike). The results demonstrate that the impedance measurement method and refined experimental conditions are suitable for further development of electrochemical biosensors for serological diagnosis of COVID-19 and/or confirmation of successful vaccination against SARS-CoV-2.

Figure 2. Schematic representation of experimental stages. pCAGGS (Creative Biogene, cat. no. VET1375) is involved in the preparation of the rSpike protein. (Drobysh M, et al., 2022)

Hamster CHO cells were used to secrete rSpike protein. This gene encodes the SARS-CoV-2 Spike extracellular domain, including amino acids (aa) 1–1208. The gene was integrated into the expression vector pCAGGS (Creative Biogene, catalog number VET1375) through the restriction sites NotI and XhoI added correspondingly at the 5' end and 3' end of the gene. These expression constructs contain the following: (i) full-length rSpike ectodomain (aa 1–1208) without transmembrane and cytoplasmic aa, (ii) furin cleavage site 'RRAR' mutated to 'GSAS', (iii) C-terminal GSN4 trimerization motif fused to the protein sequence, (iv) thrombin cleavage site. Two mutations (K986P and V987P) were introduced into the rSpike sequence to stabilize the trimer in the prefusion conformation.