Figure 5: Schematic structures of the vector and gene encoding the constructed chimeric GPI-modified anti-protein analyte scFv engineered for its expression in High FiveTM cabbage looper cells. The pIZT/V5-His vector used harbours two open-reading frames under the expression control of the Orgyia pseudotsugata multicapsid
nucleopolyhedrosis virus immediate-early 2 transcription promoters (P) and SV40 transcription terminators (T), located upstream (P) and downstream (T), respectively, of either the scFv or the chimeric construct (GFP-Zeo). The GFP-Zeo construct is built of the cDNA for a modified green fluorescent protein (GFP) fused in-frame to a cDNA that mediates resistance toward the synthetic antibiotics Zeocin (Zeo) and operates as a selection marker for the InsectSelectTM System. Insect cells equipped with the plasmid are assumed to express the two open reading frames coding for GFP-Zeo and chimeric scFv polypeptides with similar efficacies. Thus, measurement of the GFP fluorescence associated with the insect cells can be used to monitor their transfection efficacy. Furthermore, selection for Zeocin resistance will enable the identification of insect cells stably transfected with the vector and in course continuously producing the chimeric GPI-modified scFvs. In addition, the positions of the multiple cloning restriction sites as well as of the bacterial origin of replication (Ori) and of the ampicillin resistance marker (Am) within the vector are indicated. The GPI-modified scFv chimeric gene is composed (in 5’ to 3’ direction) of the secretory signal sequence I derived as a chimeric construct from rat growth hormone and somatotropin (SI), of the sequences for the К light chain variable domains (VK), for the linker (L), for the heavy chain variable domain (VH), for the histidine tag (H6) and for the influenza hemagglutinin epitope (HA) and of the GPI signal sequence II derived from authentic human placenta alkaline phosphatase (SII).