Utilization Of Tobacco For Expression Of Recombinant Bovine Chymosin And Human Epidermal Growth Factor

Plant bioreactor utilizes plant cells as platform to produce human required products in the compartnents of the cells. Plant bioreactor harbords several merits compared to other bioreactors, e.g., low overall cost, safty of products and scale-up capacity.This study has made efforts to explore the utilization of transgenic tobacco paints as bioreactor to express recombinant bovine chymosin and human epidermal growth factor, and the findings achieved were listed as following:Plant transformation vectors harboring bovine (pre)prochymosin enconding genes, namely, p33cym11,p33cym12, p33cym21 and p33cym22, were constructed. The vectors differ from the signal peptide sequences and, the recmbian chymosin will be fused with a 6 xhistidine tag for simplifying the purification process subsequently.Transgenic tobacco plants were regenerated via Agrobaterium-mediated transformation with the vector mentioned above after screening. The intergration of transgene was confirmed by PCR and Southern blotting analysis, and RT-PCR analysis was employed to demonstrate the transcription of the bovine (pre)prochymisin genes.Aurum-labeled immune-strips were used to confirm the expression of selectable mareker gene, and the results indicated that the gene has expressed in all the resistant paints tested. Immunobloting assay was employed to detect the expression of recombinant bovine chymosin and the results implied that the protein has been expressed in the tested plants. Expression level of bovine chymosin was further quantified by ELISA and turned out to be 0.22%-0.52% of total soluble protein (TSP), namely,35.2-83.2 ng/g of fresh weight. The results from milk clotting assay suggested that the plant-derived recombinant bovine chymosin is of natural bioactivity.A chloroplast transformation vector, pWX-Nt02, was constructed for tobacco transformation. Three genes including reporter gene (smFGP), selectable marker gene(aadA) and gene of interest (hEGF) are involed in the vector, and of which, the hEGF was fused to the sequence of 6×histidine tag for further purification of the recombinant protein.The vector pWX-NtO2 was introduced into tobacco chloroplast by biolistic bombardment. Transgenic plants were obtained after selection and were confirmed to be homoplastomic by detection with fluorescent assay combined with molecular testings. Results from PCR and Soutern blotting comfirmed. the intergration of transgene and the homoplasmy. The homoplastic plants exhibited stable inheritance and strictly followed the maternal inheritance characteristics.Results from fluorescent assay and SDS-PAGE analysis suggested the expression of green fluorescence protein, the reporter. Data from ELISA showed that the accumulation of GFP is 22.38% TSP, i.e.,4.22 mg/g fresh weight. GFP was purified by ammonium sulfate precipitation and gel filtration chromatography methods.Immunoblotting assays confirmed the exptession of recombinant human epidermal growth factor in transplastomic plants. The ELISA result demonstrated the expreesion level was between 0.124%～0.165% TSP, namely,23.16～25.77 ng/g fresh weight. The data from cell proliferation suggested the natural bioactivity of the plant-drived recombinant hEGF issimilar to commercial products.