Cloning of Human Coronavirus NL-63 ORF3, M and E genes for antibody production
Fisher, Randall Graeme
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Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3 C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA.