title: Reverse genetic engineering of the human rhinovirus serotype 16 genome to introduce an antibody-detectable tag.
abstract: The ability to accurately detect viral proteins during infection is essential for virology research, and the lack of specific antibodies can make this detection difficult. Reverse genetic engineering of virus genomes to alter the wild-type genome is a powerful technique to introduce a detectable tag onto a viral protein. Here we outline a method to incorporate an influenza hemagglutinin epitope tag onto the 2A protease of HRV16. The method uses site-directed mutagenesis PCR to introduce the sequence for the HA antigen onto either the C or N termini of 2A protease while keeping the relevant internal cleavage sites intact. The new viral product is then cloned into a wild-type HRV16 plasmid and transfected into Ohio Hela cells to produce recombinant virus.
authors.affiliation: Centre for Research in Therapeutic Solutions, Faculty of Education, Science, Technology and Mathematics, University of Canberra, 1 Kirinari Street Bruce, Canberra, ACT, 2601, Australia, Erin.Walker@canberra.edu.au.
authors.forename: Erin J
authors.initials: EJ
authors.lastname: Walker
authors.forename: Lora M
authors.initials: LM
authors.lastname: Jensen
authors.forename: Reena
authors.initials: R
authors.lastname: Ghildyal
mesh_terms: D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D000939:Epitopes / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D006367:HeLa Cells / Q000821:virology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D059386:Reverse Genetics / Q000379:methods; D012229:Rhinovirus / Q000235:genetics* / Q000276:immunology; D065288:Serogroup; D014162:Transfection / Q000379:methods; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus