Single-Cell Cloning of H5N1 HA-Transfected CHO Cells & Protein Expression Analysis

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dc.contributor.advisorLi, Weizhong
dc.contributor.advisorZhu, Xiaoping
dc.contributor.authorRahaman, Suneha
dc.contributor.authorSun, Keston
dc.contributor.authorYang, Alicia
dc.contributor.authorDhakal, Bibek
dc.date.accessioned2025-08-18T16:11:54Z
dc.date.issued2025-07-25
dc.descriptionCell 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.
dc.description.abstractAvian 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.
dc.description.sponsorshipThis work was supported by the National Institutes of Allergy and Infectious Diseases Grant No. 5-R01-AI-146063-05 under the National Institutes of Health. Additional funding was provided by the United States Department of Agriculture and the University of Maryland. Special thanks to all members of the Zhu Lab at the University of Maryland’s Department of Veterinary Medicine for their guidance and support.
dc.identifierhttps://doi.org/10.13016/5kui-abrv
dc.identifier.urihttp://hdl.handle.net/1903/34452
dc.language.isoen_US
dc.subjectH5N1
dc.subjectProtein Expression Analysis
dc.subjectBlotting Techniques
dc.subjectImmunology
dc.subjectSingle Cell Cloning
dc.subjectTransfection
dc.titleSingle-Cell Cloning of H5N1 HA-Transfected CHO Cells & Protein Expression Analysis
dc.typeOther

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