Production of the sheep pox virus structural protein SPPV117 in tobacco chloroplasts

Abstract

Objective A chloroplast transgenic approach was assessed in order to produce a structural protein SPPV117 of sheep pox virus in Nicotiana tabacum for the future development of a plant-based subunit vaccine against sheep pox. Results Two DNA constructs containing SPPV117 coding sequence under the control of chloroplast promoter and terminator of psbA gene or rrn promoter and rbcL terminator were designed and inserted into the chloroplast genome by a biolistic method. The transgenic plants were selected via PCR analysis. Northern and Western blot analysis showed expression of the transgene at transcriptional and translational levels, respectively. The recombinant protein accumulated to about 0.3% and 0.9% of total soluble protein in leaves when expressed from psbA and rrn promoter, respectively. Plant-produced SPPV117 protein was purified using metal affinity chromatography and the protein yield was 19.67 ± 1.25 µg g−1 (FW). The serum of a sheep infected with the virus recognised the chloroplast-produced protein indicating that the protein retains its antigenic properties. Conclusions These results demonstrate that chloroplasts are a suitable system for the production of a candidate subunit vaccine against sheep pox.

Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels

“…The RB70 insertion site has subsequently been commonly used in both biotechnology and basic research (e.g. Fuentes et al ., 2016; Krech et al ., 2013; Qu et al ., 2018; Ruf et al ., 2001, 2019; Stanbekova et al ., 2021; Wurbs et al ., 2007), including studies that yield exceptionally high transgene expression levels (Oey et al ., 2009).…”

“…Potential consequences of aadA insertion in the transplastomic RB70 line are of particular interest because the same insertion site (with identical direction of transgene expression) has been frequently used for diverse applications in basic research and chloroplast biotechnology (e.g. Fuentes et al ., 2016; Krech et al ., 2013; Qu et al ., 2018; Ruf et al ., 2001, 2019; Stanbekova et al ., 2021; Wurbs et al ., 2007). Our chloroplast ribosome profiling and transcriptome analyses revealed that not a single chloroplast gene displays significantly ( P value <0.05) or substantially (log 2 fold change >1 or <−1) altered expression in RB70 plants at either the level of transcript accumulation or ribosome coverage (Figure 8).…”

“…A good example is the RB70 insertion site, which shows negligible side effects on chloroplast gene expression (Figure 8) and was successfully used in a number of transplastomic studies (e.g. Fuentes et al ., 2016; Krech et al ., 2013; Qu et al ., 2018; Ruf et al ., 2001, 2019; Stanbekova et al ., 2021; Wurbs et al ., 2007). The RB70 insertion site has also been efficiently used in co‐transformation experiments to engineer distantly located sites in the chloroplast genome, where neighbouring‐gene effects must be avoided to obtain clean mutants (Krech et al ., 2013; Torabi et al ., 2014).…”