Journal: Human Vaccines & Immunotherapeutics
Article Title: Pentameric M2e influenza vaccine candidate generates strong immunity with limited survival benefit
doi: 10.1080/21645515.2025.2610073
Figure Lengend Snippet: Designing and expression of soluble M2e-5x protein in mammalian (Expi293F) cells. (A) Schematic illustration of expression cassettes used in mammalian vectors (pcDNA3.1(1)) for protein production. (B) The elution profile of M2e-5x protein on a Superdex 200, 16/60 column shows a single well-defined peak at ~11 mL corresponding to oligomeric soluble fractions. (C) (i)12% reducing SDS-PAGE image of a purified M2e-5x protein; lane 1 contains a pre-stained ladder and lane 2 contains 10 µg of M2e-5x. (ii) Western blot image of M2e-5x protein probed with anti-M2e (14C2) primary antibody and developed with anti-mouse IgG-HRP conjugated secondary antibody using femtolucent substrate. (D) Native-PAGE profile of the purified M2e-5x protein; lane 1, molecular mass marker, and lane 2, M2e-5x. (E) The long-term stability of M2e-5x protein is assessed at different time intervals by determining ELISA-based variations in binding efficiency with anti-M2e (14C2) antibody. (i) M2e-5x storage at 4°C for 10 days, 20 days, and up to 60 days. (ii) Storage of M2e-5x at 37°C for 24 hours, 48 hours, and 72 hours.
Article Snippet: The purity and oligomeric properties of the purified proteins were confirmed on SDS-PAGE and Blue native-PAGE (Mini-PROTEAN TGXTM, Bio-Rad)., For Blue native-PAGE analysis using 4% to 15% Native-PAGE gels (Mini-PROTEAN TGXTM, Bio-Rad, Hercules, CA, USA), Native-PAGE sample preparation buffer (Invitrogen, Waltham, MA, USA), and Bis-Tris running buffer were used., Proteins were transferred from SDS-PAGE to a PVDF membrane for western blotting with primary antibodies (mouse polyclonal sera, 1:500; anti-M2e 14C2, 1:1000, Abcam), and HRP-conjugated anti-mouse secondary antibody (1:2000, Jackson) was used for detection with chemiluminescence reagents (luminol and peroxidases, G Biosciences)., ,
Techniques: Expressing, SDS Page, Purification, Staining, Western Blot, Clear Native PAGE, Marker, Enzyme-linked Immunosorbent Assay, Binding Assay
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