Single-Cell Cloning of H5N1 HA-Transfected CHO Cells & Protein Expression Analysis
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Zhu, Xiaoping
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Abstract
Avian influenza, caused by influenza Type A virus, poses significant threats to both animal and human health worldwide. Among these, the highly-pathogenic H5N1 subtype has emerged as a critical concern, causing severe global outbreaks in poultry, substantial economic loss, and ongoing zoonotic infections in humans². In this study, we generated recombinant H5N1 proteins through single-cell cloning of transfected Chinese Hamster Ovary (CHO) cells to verify protein expression. We utilized plasmids encoding influenza H5N1 protein (strain B3.13 with Bovine IgG1) and His-tagged control proteins from bovine (strain B3.13) and human (strain D1.1) origins. Cells were transiently transfected using PEI MAX reagent and subsequently expanded in single-cell-derived clones. Protein expression was screened and confirmed via dot blot and Western blot analyses. Our findings demonstrate a robust methodological framework to produce stable recombinant influenza proteins, essential for further research toward diagnostics, therapeutics, and vaccine development.
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Cell Culture & Transfection: CHO cells were seeded in 6-well plates at a density of 2×10⁶ cells/well and cultured for 18 hours. Cells were transiently transfected with 2 µg of pDNA encoding either H5N1 protein + B3.13-BoIgG1, bovine His-tagged control protein (B3.13-His), or human His-tagged control protein (D1.1-His). Transfections were done using polyethylenimine (PEI) Max at a DNA:PEI ratio of 1:6, according to standard protocols.
Single-Cell Cloning: Cells were single-cell cloned manually into 96-well plates to ensure monoclonality. Cells were maintained under standard culture conditions until sufficient confluency was achieved.
Protein Expression Analysis (Dot Blot) & Further Confirmation by Western Blot: His-tagged control proteins (B3.13-His and D1.1-His) were probed overnight at 4°C with biotin-conjugated anti-His primary antibody (1:10,000 dilution in blocking buffer). Membranes were incubated with HRP-conjugated streptavidin secondary antibody (1:20,000 dilution in 3% BSA) for 2 hours at room temperature. H5N1 fusion protein (B3.13 - BoIgG1) was blocked with 5% milk in PBST and probed overnight at 4°C with HRP-conjugated bovine IgG antibody (1:10,000 dilution). No additional secondary antibody was required. Membranes developed using Immobilon® ECL UltraPlus Western HRP Substrate.