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Introduction of recombinant antigens against Jembrana Disease Virus into plants

Singh, Meenu (2010) Introduction of recombinant antigens against Jembrana Disease Virus into plants. Masters by Research thesis, Murdoch University.

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Jembrana disease virus (JDV) is a bovine lentivirus that causes an acute severe disease syndrome in banteng cattle (Bos javanicus) in Indonesia. In order to develop a vaccine, JDV capsid (CA) protein together with transactivator of transcription (Tat) have been produced and expressed in a microbial system with limited success, but surface unit glycoprotein (SU) is difficult to produce in E.coli as it requires glycosylation and post-translational modification. In an approach to overcome the limitations of a microbial antigen expression system, in this study, JDV fused CA-Tat and SU regions were introduced into tobacco plants and analysed for protein expression.

Plants have considerable potential for the production of biopharmaceutical proteins and peptides because they are readily transformed and can provide a cheap and reliable source of recombinant proteins. The protein synthesis pathway is highly conserved between plants and animals. Plant-derived biopharmaceuticals can also be scaled up for mass production, and are potentially safer, that is, they are not liable to be contaminated by mammalian viruses compared with products derived from animals or animal cell lines. The overall benefits are the range of recombinant proteins that can be produced in plants and the flexibility that is allowed in the engineering of new pharmaceutical proteins, which can be designed with plant expression in mind.

The aim of this work was to use transgenic tobacco lines as a system for production of recombinant antigens for JDV. CA-Tat and SU region of JDV genome were successfully cloned into the plant transformation vector and introduced into tobacco (Nicotiana tabacum) using Agrobacterium mediated plant transformation. Transgenic plants (T0 and T1) were successfully generated. A total of 12 T0 plant for CA-Tat and 21 for SU were obtained. Transgenic tobacco plants (T1) were examined by PCR, SDS/PAGE, western blotting, dot-blot and semi-quantitative reverse transcriptase (RT) PCR. RT-PCR analysis confirmed the presence of mRNA of the inserted gene in all the transgenic plant lines tested. However, using western blotting and Dot-blot, no protein expression was observed in the transgenic plants. Lack of protein expression might result from several factors, including difference in codon usage between the animal virus and plant cells, post-transcriptional and post-translational processing between plants and animals, or rapid degradation of any protein product if produced.

Another objective undertaken in parallel with this work was to study the potential of porcine circovirus (PCV) rep gene promoter to act as a novel promoter in plants, and which could provide freedom to operate. To achieve this aim, an expression construct was created containing the PCV promoter in frame with a gus gene, and 9 transformed tobacco lines (T0) were generated. Histochemical and fluorometric GUS assays were carried out to check the expression in transformed plants. Out of 9 transgenic lines, two of the PCV promoter-GUS lines showed a slightly higher level, but not statistically significant, GUS activity in leaves compared to untransformed plants and one line showed a slightly higher level of GUS activity in root tissues compared to a CaMV35S-gus positive control line. These results suggest that further study of this promoter, modifying its sequence more towards those present in plant nanoviruses, might lead to higher levels of expression of genes linked to this promoter.

Item Type: Thesis (Masters by Research)
Murdoch Affiliation: School of Biological Sciences and Biotechnology
Supervisor(s): Jones, Michael, Desport, Moira and Wylie, Steve
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