Transient expression of anti-VEFGR2 nanobody in Nicotiana tabacum and N. benthamiana.

In human, the interaction between vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) is critical for tumor angiogenesis. This is a vital process for cancer tumor growth and metastasis. Blocking VEGF/VEGFR2 conjugation by antibodies inhibits the neovascularization and tumor metastasis. This investigation designed to use a transient expression platform for production of recombinant anti-VEGFR2 nanobody in tobacco plants. At first, anti-VEGFR2-specific nanobody gene was cloned in a Turnip mosaic virus (TuMV)-based vector, and then, it was expressed in Nicotiana benthamiana and Nicotiana tabacum cv. Xanthi transiently. The expression of nanobody in tobacco plants were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), dot blot, enzyme-linked immunosorbent assays (ELISA), and Western blot analysis. It was shown that tobacco plants could accumulate nanobody up to level 0.45% of total soluble protein (8.3 µg/100 mg of fresh leaf). This is the first report of the successful expression of the camelied anti-VEFGR2 nanobody gene in tobacco plants using a plant viral vector. This system provides a fast solution for production of pharmaceutical and commercial proteins such as anti-cancer nanobodies in tobacco plants.

Bioinformatical evaluation of desiccation-responsive rd29A gene in Arabidopsis thaliana.

In Arabidopsis thaliana expression of two Desiccation-Responsive rd29 genes are intensity induced under drought, salinity and temperature (heat and cold) conditions. These two genes called, rd29A and rd29B are located in 8-9 Kb of Arabidopsis genome. Also, transgenic plants containing these genes have more tolerant toward environmental stresses. Since rd29A has an important role in various non-biological stresses, in this study genetic and promoter analysis of this gene was performed, rd29A nucleotide sequence was obtained from NCBI gene bank, for bioinformatic analysis used the Map viewer, Plant care and Genevestigator software. In Arabidopsis rd29A has 8048 nucleotides and encode a linear mRNA with 2133bp. It located at AT5G52310 locus on chromosome 5 between nucleotides 21241898 and 21242457 and contains 4 exons and 3 introns. Promoter region analysis to determine the gene regulatory elements performed with using 1500 bp of the 5 'UTR sequence and the results indicated that the existence of diverse regulatory elements such as ABRE, DRE, Box I, C-repeat/DRE and TCA-element which represent involvement of this gene in different physiological pathways in plants. In addition the TATA-Box was identified as Core promoter in -30 position. Also, the pattern of gene expression was examined by Microarrays data and it was found that the expression levels were variable in different developmental stages, so that the lowest and highest expressions were in germination and mature stages. It seems that using of this gene promoter in expression cassettes can raise tolerance of transgenic plants to environmental stresses.