Bibliography: leaves 126-145. / Expression of the nefgene from HIV-1 subtype C in Nicotiana benthamiana was carried out using a TMV -based vector with the aim of developing a plant-based candidate vaccine for HIV-1. The nef gene of the DU151 isolate of mV-l subtype C taken from a recently seroconverted individual was amplified by PCR with a deletion of 59 amino acids from the cytotoxic N-terminal. The amplified gene was inserted into a bacterial expression vector pProEXHTb for rapid expression of Nef protein, which was used as a diagnostic tool in the development of an indirect ELISA assay for detection of Nef in Nicotiana benthamiana. An indirect ELISA assay was developed using a commercially available polyclonal anti Nef antiserum raised in sheep. The role of codon optimization in expression of Nef in benthamiana was investigated. A synthetic nef gene was constructed based on the codon usage of benthamiana. The plant codon optimized gene and the wild type nef genes were inserted into the TMV -based vector pBSG1057. RNA transcripts from both constructs were used to infect young benthamiana plants. Expression of nefmRNA was confirmed by RT -PCR analysis of total RNA extracted from plants inoculated with respective constructs. The Nef protein was expressed at low levels which were detectable by ELISA. Nef was detectable by Western blot after concentration of plant extract using a membrane filter device. Quantitative analysis of Nef expression in plants was done by western blot on concentrated plant extract from three separate infections. Codon optimization of the nef gene improved the expression of Nef by a factor of about two.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/4242 |
| Date | January 2003 |
| Creators | Bandawe, Gama |
| Contributors | Rybicki, Ed |
| Publisher | University of Cape Town, Faculty of Science, Department of Molecular and Cell Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
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