In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays
Section snippets
Target-primed plasmid amplification
Target-primed plasmid amplification as described previously [10] was used throughout. Briefly, megaprimers for target-primed plasmid amplification were amplified using a DeepVent (New England Biolabs) proofreading polymerase and primers (Biomers) of 60 nt length (detailed primer information is available on request). Amplified megaprimers were column- or gel-purified (Illustra DNA purification kit, GE Heathcare; QiaEx Band extraction kit, Qiagen). Megaprimers were used in concentrations of at least
Construction of recombinant baculoviruses
Initial attempts to express full-length IAV HA in insect cells failed because the recombinant antigen was trapped in insoluble intracellular deposits from which the protein could not be solved under native conditions using Triton X-100, Na-desoxycholate, Igepal Ca-630, zwittergent, Chaps, or N-dodecyl β-d-maltoside detergents (data not shown).
Therefore, alternative strategies of secretory baculovirus-based expression of a soluble HA variant were adopted and several constructs were produced (
Discussion
We have produced an array of recombinant influenza A HA subtype H5 and H7 constructs for expression in insect cells. Secretable fusion proteins that were in vivo mono-biotinylated using BirA biotin ligase-expressing bigenic baculoviruses proved to be superior in handling ease and authentic antigenicity over conventionally tag-purified and chemically biotinylated forms as a diagnostic antigen. The HA constructs were designed for primary use in serodiagnostic applications by developing a system
Acknowledgments
This study was funded by the Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV), Germany (FSI project 2-1.2.1). We are grateful to J. Stech (Institute for Molecular Biology, Friedrich Loeffler Institute, Germany) for offering his advice concerning target-primed plasmid amplification and to S. Boudko (Shriners Hospital, Portland, OR, USA) for providing the fibritin–foldon sequence. We are also grateful to the technical staff of the National Reference Laboratory for Avian
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