Virus Vector-Based Expression of Anti-VEGF Recombinant Protein in Chenopodium Quinoa Plants

Background and Aim: Pharmaceutical proteins production in plants platforms (Molecular farming technology) is a low cost and safe alternative to mammalian, bacterial and yeast cells- based platforms. Anti-VEGF (vascular endothelial growth factor) protein is a neutralizing nanobody against VEGF factor. VEGF is the most key factors in the tumor angiogenesis. Therefore, its neutralization using Anti-VEGF nanobody is proposed as alternative strategy for cancer therapy. In this study, we investigate the expression of Anti-VEGF recombinant protein in chenopodium quinoa plants using plant virus-based vector technology.Methods: Seeds of chenopodium quinoa were planted on sterilized soil (mixed with compost and perlite. The seedlings were kept in a phytotron (photoperiod condition of ۱۶ h light/۸ h and ۲۵ ֯C).After ۲ weeks, the target plants were inoculated using ۱۰ microliters (۱ µg/µl) of extracted plasmids. Recombinant viral plasmids (containing Anti-VEGF nanobody coding sequence) were extracted by the mini-prep plasmid purification kit. The inoculated plants leaves were harvested for expression analysis of the target gene at ۱۴ days after inoculation. After total RNA extraction (using RNeasy Plant Mini Kit) and cDNA synthesis, RT-PCR was performed for on cDNA using Anti-VEGF gene specific primers. Total soluble protein (TSP) was extracted from inoculated leaves and then, Dot-blot and ELISA analysis were performed.Results: The RT-PCR analysis was performed to detect of the Anti-VEGF gene transcripts in inoculated plants. The presence of the expected fragment (۳۷۸ bp) in inoculated plants confirmed the successful transcription of the Anti-VEGF gene. Dot-blot assay indicated that the plant-derived Anti-VEGF protein was recognized by specific antibodies against protein and developed brown color. No brown color spot was observed in control plants. ELISA test was used to quantify the amount of Anti-VEGF protein in TSP. Anti-VEGF protein was identified using its specific antibody. After performing this test, there was a significant difference between the optical absorption (۴۵۰ nm) of the protein extracted from the inoculated plants with viral vector and the protein extracted from the control plant.Conclusion: This Study was the first report of the successful expression of the Anti-VEGF protein in chenopodium quinoa plants using virus-based vector technology. This technology provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in chenopodium quinoa plants.

Keywords:

Recombinant protein , Molecular farming , Angiogenesis , Nanobody , Viral vector