Journal: bioRxiv

Article Title: A broadly protective CHO cell expressed recombinant spike protein subunit vaccine (IMT-CVAX) against SARS-CoV-2

doi: 10.1101/2023.04.03.534161

Figure Lengend Snippet: (A) is the viability and viable cell density (VCD) of ExpiCHO cells transfected with the pcDNA3.4–IMT–C20 construct during transient production. (B and C) are the SDS-PAGE and Western Blot, respectively, of the day-wise samples of transfected ExpiCHO cells from Day 3 to Day 11. (D) IMT-C20 cloning in Freedom pCHO 1.0: The gene (IMT-C20) was amplified from pcDNA 3.4 using gene-specific primers designed such that the forward and reverse primers had sites for AvrII and PacI restriction enzymes, respectively. The amplified gene was purified and digested using AvrII and PacI. Freedom pCHO 1.0 intact vector was also double-digested using the same enzymes (AvrII and PacI) and the larger fragment was extracted from 1% agarose gel. IMT-C20 was ligated in Freedom pCHO 1.0 and was immediately used to transform chemically competent E. coli DH5α cells. Freedom pCHO 1.0–IMT-C20 construct was isolated from the transformed E. coli cells and was double-digested with AvrII and PacI to confirm the ligation. Lanes 2 and 3 in the agarose gel image depict the undigested and the digested Freedom pCHO 1.0– IMT-C20 construct, respectively.

Article Snippet: The gel-purified IMT-C20 and the empty Freedom pCHO 1.0 vector were double digested using AvrII and PacI (NEB, USA) restriction enzymes following the manufacturer’s instructions and were resolved on 1% agarose gel.

Techniques: Transfection, Construct, SDS Page, Western Blot, Clone Assay, Amplification, Purification, Plasmid Preparation, Agarose Gel Electrophoresis, Isolation, Transformation Assay, Ligation