<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by  / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)  / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a  / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in  / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of  / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from  / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified  / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie  / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the  / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the  / Western Cape.</p>
| Identifer | oai:union.ndltd.org:UNWC/oai:UWC_ETD:http%3A%2F%2Fetd.uwc.ac.za%2Findex.php%3Fmodule%3Detd%26action%3Dviewtitle%26id%3Dgen8Srv25Nme4_7918_1375971791 |
| Date | January 2012 |
| Creators | Mnyamana, Yanga E. |
| Source Sets | Univ. of Western Cape |
| Language | English |
| Detected Language | English |
| Type | Thesis and dissertation |
| Format | |
| Coverage | ZA |
| Rights | Copyright: University of the Western Cape |
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